Microscopy tools for product innovation

Abstract

Food Science and TechnologyVolume 33, Issue 4 p. 51-55 FeaturesFree Access Microscopy tools for product innovation First published: 13 December 2019 https://doi.org/10.1002/fsat.3304_14.xCitations: 3AboutSectionsPDF ToolsExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat Mark Auty of RSSL explains the different types of microscopy used to study food ingredients and products and assesses their performance in different applications. Introduction Consumer quality of any food product is strongly influenced by the three-dimensional structural arrangement of the ingredients. Knowing the chemical composition and bulk physical properties of food may not be enough to explain processing effects or consumer perception; how the individual ingredients are dispersed and how they interact at the molecular, nano- and micro-length scales is key. Food microstructure analysis provides a direct link between physicochemical properties, process behaviour and organoleptic qualities (Figure 1, page 52). Microstructure employs a wide range of imaging tools, mostly microscopes, that each give valuable insights into product behaviour as well as providing an essential investigative toolkit to understand product failure modes. Figure 1Open in figure viewerPowerPoint Diagram showing inter-relationships between microstructure and functionality of food products Imaging techniques Microscopy techniques used to study food microstructure were originally adapted from either biology or material science disciplines and were restricted to optical or electron microscopy. However, over the past 20 years, food microstructure has been increasingly recognised as important in food science and now there are a wide range of imaging tools and methodologies being applied to food research1. Many foods have high levels of moisture, fat or sugar and preserving the original microstructure of such materials may be difficult, particularly for electron microscopy that may require low moisture, conductive specimens. Dried ingredients, such as spray dried powders, crystalline sugars, starches etc. with a moderately small particle size (<100μm) may be examined by optical microscopy in their natural state and require little sample preparation. Highly refractile or opaque solid and semi-solid food materials, however, need to be rendered thin enough to transmit light and generally this is achieved either by compression or sectioning. Soft materials may be compressed on a microscope slide. Solid food materials and cellular tissues may be chemically fixed, dehydrated then embedded in paraffin wax or plastic resin prior to sectioning by microtomy. Alternatively, frozen sections, approximately 5-20μm thick, may be cut in a cryostat. Sectioned material can then be observed using light microscopy. Powdered ingredients, for example spray-dried milk powder particles, should be mounted in a clear, immiscible viscous liquid, such as sunflower oil, to improve resolution and restrict Brownian movement. The main microscopy techniques used in food research are given in Table 1. Table 1. Main techniques used in food microscopy Technique Radiation type Approximate resolution Application Stereo-microscopy Photons 5μm Overview of microstructure, large pores Light microscopy Photons 200nm Ingredient localisation, crystallisation, particle shape & size Confocal microscopy Photons 200nm Ingredient localisation, 3D information Scanning electron microscopy Electrons 4nm Large depth of field – simulated 3D view, high resolution (nano-scale) Transmission electron microscopy Electrons 1nm Fine structural detail, macromolecular interactions Atomic force microscopy N/A (physical cantilever) <1nm Surface topology, nano-mechanical behaviour X-ray Microtomography X-rays <1-10μm Non-destructive 3D structure based on atomic contrast Over the past 20 years, food microstructure has been increasingly recognised as important in food science and now there are a wide range of imaging tools and methodologies being applied to food research. Conventional optical microscopy There are several optical microscopy approaches that can be used for studying different types of food materials with different characteristics and properties: Bright field Bright field illumination employs an axial cone of light from the condenser, which is transmitted through the specimen and is commonly based on Köhler illumination. This technique is useful for high contrast specimens, for example in coloured food particles. Stains or dyes may be used to impart colour to the specimen. Typical food stains for food products include Iodine/potassium iodide to stain starch, toluidine blue to stain proteins and polysaccharides and Sudan Red to stain lipids. Polarised light A polarised light microscope consists of two polarising plates arranged perpendicularly, one below the condenser (the polariser) and a second above the objective (the analyser). If the sample is isotropic, incident polarised light is not rotated, and no light is transmitted. If the polarised light passes through an anisotropic substance, such as a lactose crystal, part of the light is rotated and passes through an analyser appearing bright (birefringence). Polarised light microscopy is very useful for characterising starch gelatinisation or crystallisation, such as lactose crystallisation in spray dried milk powders (Figure 2). Figure 2Open in figure viewerPowerPoint Spray dried skim milk powder particles a) fresh powder, b) powder stored at 55% relative humidity for 24 h. Polarised light micrographs taken using partially uncrossed polarising filters, reveal extensive birefringent lactose crystals (b) while allowing visualisation of particle shape and occluded air bubbles (dark circles), Scale bar = 100μm. Phase contrast This technique has traditionally been used to study transparent materials and can be useful for characterising food emulsions or bacteria in yoghurt. Differential interference contrast This technique requires the addition of specialised optical elements to a basic light microscope setup but gives excellent results and has superseded phase contrast for studying transparent materials. A polariser and prism are located above and below the specimen, respectively and differences in refractive index are visualised in relief. This technique is particularly useful for studying fat droplets in milk or phase separation in transparent/translucent systems, such as ice cream mix. Confocal Scanning Laser Microscopy Confocal scanning laser microscopy (CSLM) is a form of epi-fluorescence microscopy. Conventional fluorescence microscopy employs ‘wide field’ illumination where the volume of sample above and below the plane of focus is uniformly and simultaneously illuminated. The key feature of confocal imaging is that both the illumination and detection systems are focused simultaneously on a single volume element in the specimen, achieved by positioning a pinhole close to the detection source. This requires thin, relatively transparent, samples but results in out-of-focus blur that reduces resolution and specimen contrast. CSLM employs a diffraction-limited spot, which is detected by the pinhole placed in front of the emitted light detector greatly reducing out-of-focus information. CSLM is now widely used in food microstructure research. CSLM has many advantages which include: 1. Three-dimensional imaging by ‘optical’ sectioning and digital reconstruction, 2. Sub-surface imaging minimises sample microstructure disturbance, 3. Improved resolution over conventional optical microscopy, 4. Simultaneous detection of two or more ingredients via multiple fluorescent probes, 5. Dynamic processes can be studied under controlled environmental conditions using appropriate sample stages and fast acquisition rates. The technique remains diffraction limited, with a maximum resolution of ~200nm and the sample needs to be relatively flat. The ability to clearly visualise internal food microstructure gives unique insight into its true three-dimensional arrangement and allows for simple differential fluorescent labelling of specific components such as fats or proteins (Figure 3 for examples). Figure 3Open in figure viewerPowerPoint Confocal scanning laser micrographs of various dairy products labelled with fluorochromes. b, c, d and f are dual labelled with Nile Red and Fast Green FCF to show fat (green) and protein (red), respectively. a) whey protein-stabilised emulsion labelled with Rhodamine B (pseudo-coloured green) to visualise protein at the droplet interface, scale bar = 10μm. b) Full-fat dairy yoghurt showing protein network and small fat droplets in addition to pores (dark regions), scale bar = 10μm. c) Mayonnaise showing fat droplets and discrete aggregated protein particles, scale bar = 75μm. d) Cheddar cheese showing irregular shaped fat pools and continuous protein matrix, scale bar = 25μm. e) Butter labelled with Nile Red (greyscale image) showing characteristic rounded butterfat crystals (dark grey, arrowed), water droplets (dark circles) and continuous fat phase (bright), scale bar = 5μm. f) Fat-filled whey protein enriched dairy powder showing fat droplets entrapped in continuous protein matrix, scale bar = 5μm. Electron microscopy (EM) Electron microscopes comprise an electron emitter encased in a vacuum. Accelerated electron beams have a much shorter wavelength and consequently greatly increase resolution compared to light radiation. The electron beam is focused with electromagnets and an image is produced either by passing the beam through a thin section of material as in transmission electron microscopy (TEM), or by electrons striking the surface of a bulk sample and emitting electrons as in scanning electron microscopy (SEM). SEM has been used extensively in the study of food ingredients and products. Traditionally, chemical fixation and dehydration protocols were necessary to preserve biological specimens from the harsh environment of electron microscopes. Interpretation of EM images requires a thorough understanding of the effects of sample processing on the integrity of microstructural elements and the possible generation of artefacts. TEM involves passing a narrow beam of electrons through a thin specimen prepared either as a negatively stained dispersion or in the form of a thin section or a metallic replica. For resin-embedded thin sections, sample preparation can be extensive and usually involves chemical fixation, solvent dehydration and embedding in a polymer-based resin. Ultrathin sections ~90–150nm thick are cut using a glass or diamond knife with an ultramicrotome. The sections are post-stained to increase contrast and carbon coated to prevent beam damage. In SEM, secondary electrons emitted by the sample provide topographic information with a high depth of field. Conventional SEM has been extensively used to characterise the surface morphology of low moisture products including dairy powders (Figure 4) or snack products, such as crackers or confectionery. Figure 4Open in figure viewerPowerPoint Scanning electron micrographs of spray dried milk derived powders. a) Milk protein concentrate primary particles showing smooth surfaces with fine particles, scale bar = 2μm. b) Infant milk formula spray dried powder, showing agglomeration of primary particles and fines into large particles. Scale bar = 50μm. Images taken at 2kV accelerating voltage. Modern SEM instruments may be fitted with field emission electron sources which allow for much lower voltages (0.1–5kV) thereby reducing charging effects. Cryo-SEM is particularly useful for food products and involves rapid freezing of small samples, fracturing under vacuum and then transferring to the SEM chamber onto a special cryo-stage, where frozen hydrated bulk samples can be directly imaged under the electron beam. Cryo-SEM has been successfully used to study dairy spreads, mayonnaise and ice cream. Freeze fracturing allows visualisation of internal structures, such as air, protein or and fat distribution in whipped cream, cheese and yoghurt (Figure 5). Figure 5Open in figure viewerPowerPoint Cryo scanning electron micrographs of various dairy product images at -125°C after freeze fracturing a) whipped cream showing the interface between two air bubbles (A) that contains fat droplets (arrow) and milk proteins dispersed in the aqueous phase. b) Cheddar cheese showing fat globule (F), (P) and streptococci-like bacteria at the fat/protein interface (arrow). c) Full-fat yoghurt showing starter bacteria (arrows) and fat droplet partially embedded in the protein matrix. d) Full-fat yoghurt cryo-fractured fracture showing large pores caused by ice crystals forming during freezing (arrows). Scale bar = 1μm. A major challenge with conventional EM techniques is preventing damage to nonconductive specimens caused by the high-energy electron beam. Biological materials, being relatively non-conductive, are especially susceptible to beam damage and require coating with a conductive layer of carbon or heavy metal. In addition, the electron beam requires a vacuum to prevent dissipation of electrons in the column. Variable pressure (sometimes termed low vacuum mode) or true ‘environmental’ scanning electron microscopes (ESEMs) overcome both specimen-charging effects due to the electron beam and the need for dehydration by allowing a gas of 1–20 Torr within the imaging chamber. The gas may be inert or water vapour, consequently samples may be observed at saturated water vapour pressure in the case of true ESEMs. Variable pressure SEM employs a weak vacuum that still allows direct observation of samples with moderate moisture contents, such as cheese or meat products. Dynamic CSLM techniques – making and breaking food structures Food processing is not a steady-state process and many foods undergo several transformations during manufacture, storage and consumption. These processes often include mixing, heating, pH adjustment or complex biochemical transformations, such as fermentation. To understand how unit operations affect microstructure and ultimately behaviour of a food product, microscopy images are taken at key process steps for correlation with bulk physical measurements, such as viscosity or particle size. These ‘snapshots’ along the process line cannot, however, show what is happening during a fast-moving process, such as shearing, product aggregation or breakage, so dynamic imaging techniques are needed. Dynamic CSLM allows direct monitoring of food transformations, such as milk gelation for yoghurt or cheese manufacture (Figure 6). Using this technique, we can follow the particle movement, aggregation and subsequent network formation of casein micelles during real-time acidification at controlled temperature. Figure 6Open in figure viewerPowerPoint Confocal scanning laser micrographs taken from time lapse series of skim milk acidified with glucono-delta lactone at 40°C, protein labelled with Nile Blue (bright areas). Scale bar = 25μm. Conversely, food is designed to break down during consumption, but little is known of how fracture behaviour is affected by microstructure. Micro-tensile stages are now available for studying deformation of solid food materials at the microscopic scale. An example is shown in Figure 7 where two fat-filled protein gels were fractured to characterise the movement and release of liquid fat during breakage. This type of study highlights the importance of microstructure on the fat release properties of food and how new reduced-fat products can be designed to optimise fat release and sensory properties. Figure 7Open in figure viewerPowerPoint Confocal scanning laser micrographs of fat-filled whey protein gels monitored using a micro-tensile stage. Protein is pseudo-coloured red, fat pseudo-coloured yellow/green. A1-A5 gelled at pH7.0 and is fine stranded, B1-B5 is gelled at pH5.4 and is particulate. Notch propagation is vertical and fracture properties measured in extension. Note deformation (circle) and de-bonding (arrow) of fat in the particulate gel which contrasts with fracture through fat droplets for fine-stranded gel. Scale bar 25μm. Emerging microscopy techniques There is a need to develop new imaging tools that are relevant to the metastable complexity of dairy products. These new tools face the following constraints: Speed of acquisition: many dairy products are highly metastable or liquids. Chemical mapping: particularly for complex organics, such as proteins. High resolution: spanning the nano-micro length scales. Controlled environment: for example, temperature or humidity. ‘Microscopy has the advantage that it is a direct technique allowing visualisation of reality rather than an assumed structure derived from bulk physical measurement.’ New developments in microscopy and imaging are beginning to address these issues and are discussed below. Atomic force microscopy Scanning probe microscopies are a group of techniques originally designed to characterise surfaces at the atomic scale. Atomic force microscopy (AFM) is one such technique which allows imaging of soft composite materials and liquids at molecular scales with a resolution of <1nm. AFM is increasingly being applied to food materials including chocolate and dairy products. A cantilever with a needle-like probe tip is scanned close to or touching the sample surface and deflection monitored by a laser and photodiode and converted to topographical and/or nano-mechanical information. Atomic force microscopy has been used to study polysaccharides, starches and food proteins. Chemical mapping – confocal Raman microscopy The development of confocal Raman microscopy will become a key tool in the microstructural characterisation of food ingredients and products. Raman imaging is a powerful technique for generating detailed chemical images based on a sample's Raman fingerprint spectrum. A complete spectrum is acquired at each pixel of the image, which is then processed to generate false colour images based on material composition, phase, crystallinity and strain. This allows for 3D molecular mapping of a material with a spatial resolution of <1μm. Since there is only one Raman photon for every ~106 fluorescence photons, care is needed to avoid natural fluorescence which can swamp the weak Raman signal. For spectral mapping, acquisition and processing times are also much slower than those of traditional confocal microscopes and can take several minutes or hours rather than seconds. Recent developments in technology have reduced the time to acquire a full spectrum down to just under a millisecond. Despite these limitations, early studies demonstrate the power of the technique for localisation of specific plant biopolymers, phenolic compounds, fat crystallisation and polymorphism. Non-destructive 3D imaging – X-ray computed microtomography (XMT) XMT is a non-destructive analysis technique used to visualise and characterise objects in three dimensions. It is the process of imaging an object from many directions using penetrating radiation, such as X-rays, then using a computer to determine the interior structure of that object from these projected images. It has been used to probe the structure of dairy products, such as cream cheeses and yoghurts, in addition to visualising the microstructure of loose-packed and compacted samples of spray-dried skim milk powder and whole milk powder and to quantify the proportion of both interstitial and occluded air voids in each sample. Individual beam scans can be analysed and reconstructed for true volumetric morphological analysis. Disadvantages include the lack of atomic number contrast in many food materials and the relatively slow acquisition speed (minutes or hours). Newer high-resolution instruments are now available that can image samples at the nanometre scale. New phase contrast imaging and faster data processing rates will further improve XMT making it a very powerful technique for direct imaging of food products in their bulk state and at sub-micron resolution in full 3D. Measuring – image analysis Microscopy has the advantage that it is a direct technique allowing visualisation of reality rather than an assumed structure derived from bulk physical measurement. Image analysis can be used to measure features of an image and there are several software options available, both open source and commercial packages. Techniques, such as CSLM and XMT, are particularly suitable for image analysis due to the clear discrimination of the major phases and accurate 3D structure. Particle/droplet size, shape, pore size or linearity, connectedness, clustering etc. are all measurable using basic software. The advent of machine learning and artificial intelligence will allow more complex vision-based analysis from large 3D/4D image datasets. Conclusions Microscopy offers a powerful set of tools to help understand the complex relationships between structure and function in food products. There are now a wide variety of microscopy techniques to choose from with new techniques emerging. It is important to know the strengths and weaknesses of each technique and use a correlative approach to ensure correct interpretation of images for each application. Dr Mark Auty, Research Principal, Food Microstructure, Reading Scientific Services Ltd Reading Science Centre, Whiteknights Campus, Pepper Lane, Reading, RG6 6LA, UK Email Mark.Auty@rssl.com Telephone +44 (0)118 918 4225 Web rssl.com REFERENCES 1Zafeiri, I., Wolf, B. 2019. Sustainable Pickering emulsions. Food Science and Technology 33(3): 20- 42 Citing Literature Volume33, Issue4December 2019Pages 51-55 FiguresReferencesRelatedInformation