Guest Food Microscopists 1

H. Douglas Goff, Ph.D.

Professor of Food Science Dr. Doug Goff has studied a great variety of dairy foods at the Department of Food Science, University of Guelph in Ontario, Canada. One of the foods which he examined by microscopy, is ice cream. It is very popular with consumers but has received little attention from the microscopists. Dr. Goff used cryo-SEM and TEM to produce the micrographs below, and he provides us with the following description of what he sees.

TEM of 3 fat globules (orange) showing only little crystallinity (lighter lines) inside the globules. Proteins can be seen as black spheres.

TEM of 1 large and 1 small fat globules (orange) showing their contents almost completely crystallized (lighter lines inside the globules).

Ice cream structure under the microscope is a marvelous thing to see. While most consumers see it as a cold, creamy, smooth, delicious dessert, it is no easy task to produce and maintain a structure that will deliver these attributes! Information on the manufacture of ice cream can be found at the University of Guelph - Dairy Science and Technology Education website . Briefly, the first step of ice cream manufacture is to combine the ingredients (cream, milk, milk solids, sugars, and <0.5% of stabilizers and emulsifiers) into a mix, which is pasteurized and homogenized. This creates a milkfat emulsion, comprised of millions of tiny droplets of fat dispersed in the water phase, each surrounded by a membrane of proteins and emulsifiers. The proteins can be seen in the top image as the black spheres adsorbed onto the fat globule surface. The sugars, also added to the mix during processing, are dissolved in the water phase. After the mix is cooled, the milkfat partially solidifies (as does butter when you cool it), so that each droplet consists of solid fat crystals cemented together by liquid fat. The two images at left, produced by TEM, show low crystallinity (top image) and a high crystallinity (lower image) of fat droplets in an ice cream mix.

The ice cream mix is then whipped and frozen, a process that creates two more discrete structural phases, millions of tiny ice crystals and air bubbles dispersed in the concentrated unfrozen mix. The water, which comes from the milk or cream, freezes into ice, and the dissolved sugars become increasingly concentrated in the unfrozen phase as more ice forms. Ice crystals should be 30-50 µm in diameter - the larger they are from manufacture or become due to temperature fluctuations in storage, the more coarse/icy the ice cream will taste. The whipping processs helps keep the ice crystals small and discrete. The colour image at left, produced by cryo-SEM, shows a cross section of frozen ice cream, illustrating the four microscopic phases of frozen ice cream: ice crystals (blue - 'C'), air bubbles ('A'), fat droplets ('F' - for details see the micrograph at right), and the unfrozen phase ('S' - yellow).

The whipping process also helps to incorporate air in the form of tiny bubbles 50-80 µm in diameter. Approximately one half of the volume of ice cream is air (without it, ice cream could not be scooped or chewed in the mouth), but the fact that it is dispersed in tiny bubbles means that the ice cream tastes smooth and the air is not noticeable. All of the fat droplets play an important role at the air interface, helping provide that smoothness. The process of freezing and aeration of the mix causes the milkfat emulsion to undergo a process called partial coalescence, in which the fat droplets form clusters and aggregates of fat that surround and stabilize the air bubbles. This same process is what creates structure in whipped cream (the structure of the fat and the air in whipped cream and ice cream are very similar). The colour image at right, also produced by cryo-SEM, shows another cross section of frozen ice cream, illustrating an air bubble lined with the agglomerated fat and individual droplets (yellow).

The next time you enjoy a cone of ice cream, pause for a moment and marvel at its structure!

To learn how frozen ice cream can be embedded in a resin to be sectioned and subsequently examined by transmission electron microscopy please read on:

A great deal is known about ice cream structure and the contribution of fat to that structure, through fat destabilization, but one element of structural information that has been missing is the nature of fat structures within the frozen system itself, and the interaction of these fat structures with air. It has not been clear whether the appropriate schematic model for destabilized fat networks is one of partially coalesced (clustered) fat globules adsorbed to air bubbles, or fat clusters primarily in the bulk, or both. The role of coalescence in fat destabilization is not clear. There has been debate about the presence of liquid fat at the air interface, the origin of which might be non-crystallized triglyceride escaping from the fat globule during rupture. It has also not been clear what happens to the structure of fat at air bubble interfaces during the process of expansion of the bubbles, which must happen in the transition from imposed back pressure to atmospheric pressure upon drawing ice cream from the swept-surface freezer.

What was thus needed was an electron microscopic technique that would allow the visualization of fat and air structures in the natural frozen state. The first reported electron microscopy methods for the study of ice cream structure utilized thin-section transmission electron microscopy (TEM) of highly fixed samples of melted ice cream, or freeze-fracture, metal shadowing, and examination of replicas of frozen ice cream with TEM. The development of low temperature (cryo) scanning electron microscopy (LT-SEM) has allowed further detailed studies of ice cream structure, particularly the ice phase. However, both the metal replication TEM technique and LT-SEM are limited to the features of fractured surfaces, and in the latter case, magnification and resolution are not high enough to discern interactions between fat globules. Thin-sectioning for TEM after sub-ambient temperature fixation has been used to view ice cream mix and melted ice cream, however, the use of aqueous fixatives has limited the use of thin-section techniques to non-frozen samples. We have recently used a TEM thin-section method through application of a freeze-substitution technique for sub-zero sample fixation. There has been no reported use of cryo- sectioning of ice cream for viewing with cryo-TEM, but this may be forthcoming with the presence of new, sophisticated instrumentation, and may provide the ultimate in structural information.

In the freeze-substitution TEM method, ice cream specimens were taken from the inner bulk of the hardened samples at -25¡C with a surgical blade and immediately placed into liquid nitrogen (-196¡C), where they were broken into <1.0 mm³ pieces with the surgical blade under slight force. Frozen specimens of ice cream were transferred into vials that contained a mix of fixatives consisting of 2.15 % (w/v) uranyl acetate, 3.23 % (v/v) glutaraldehyde and 1.0 % (w/v) osmium tetroxide in absolute methanol, which had been kept at -196¡C in liquid nitrogen (the fixative mixture is solid at this temperature). The vials were then placed in a -80¡C freezer for 4 days, where after warming up from -196¡C to -80¡C the fixative mixture melted and the gradual freeze-substitution of ice with methanol took place. Samples were then transferred to -40¡C for 1 day where some fixation with glutaraldehyde, osmium tetroxide and uranyl acetate took place (there was evidence of staining after treatment at -40¡C). Samples were then transferred to -20¡C for 2 days where fixation proceeded at a faster rate and further staining was accomplished. Then the fixative mixture was replaced by washing the specimens in precooled (-20¡C) 100% methanol followed by repeated washing (3x) with absolute ethanol. Specimens were placed into gelatin capsules and the infiltration with low temperature embedding resin Lowicryl HM20 (Marivac Ltd., Halifax, NS, Canada) was performed in the following way: one wash with ethanol:Lowicryl 1:1 at -20¡C for 10 min, one wash with ethanol: Lowicryl 1:3 at -20¡C for 10 min and three washes with 100% Lowicryl at -20¡C. The resin-infiltrated samples were polymerized under 360 nm UV light at -20¡C. Resin blocks were sectioned at a thickness of 90 nm using an LKB ultramicrotome. Sections were mounted immediately on formvar-coated grids (Marivac Ltd., Halifax, NS, Canada). No post-staining was carried out, as the applied fixation process provided a sufficient contrast for TEM imaging. The sections were observed and photographed in a TEM at 75 kV.

Figures 1 and 2 are cross sections of ice cream at low magnification as viewed by TEM after freeze substitution, showing the unfrozen serum phase (s), air bubbles (a), and an ice crystal (i). In the unfrozen serum, dispersed fat globules and casein micelles are just discernible. At higher magnification (Figures 3 and 4), fat globule (f) adsorption to the air serum interface (a) and fat clustering (fc) from partial coalescence in the serum phase can be seen. Highly freeze-concentrated casein micelles (c) can also be seen in the serum phase.

What was concluded from a detailed study of ingredient and process variables was that the structures created by increasing levels of fat destabilization in ice cream were observed as an increasing concentration of discrete fat globules at the air interface and increasing coalescence and clustering of fat globules both at the air interface and within the serum phase. However, air interfaces at the highest levels of fat destabilization were not completely covered by fat globules, nor was there evidence of a surface layer of free fat. Air interfaces from continuous and batch freezing were similar.

Reference:
Goff, H. D., E. Verespej, and A. K. Smith. 1999. A study of fat and air structures in ice cream. International Dairy Journal 9:785-797.

Dr. A. K. Smith has had a long career as a food electron microscopist at the University of Guelph. In her Ph.D. thesis she studied the microstructure of whipped cream. As the previous contribution by Dr. H. D. Goff has shown, Dr. Smith also participated in the studies of ice cream. She too used freeze substitution to obtain TEM micrographs of whipped cream to reveal its microstructure depending on preliminary processing of the cream. To calculate dimensions of the air cells in whipped cream, Dr. Smith used the methods of stereology in conjunctions with SEM and has described them below.

"Stereology" is defined as "the knowledge of space" (Rhodes and Khaykin, 1986). This knowledge allows three-dimensional morphological features to be statistically characterized from geometrical two-dimensional data (Rhodes and Khaykin, 1986). Stereology is a fast and efficient method for generating data from (SEM) images which do not lend themselves well to a more automated image analysis approach.

The problem inherent in SEM images is the fact that the leading edge of a feature is closer to the secondary electron detector and therefore, appears brighter. As a result, boundaries are not defined by specific, uniform grey levels making it difficult, if not impossible, to isolate critical image features from the background through image processing techniques. Instead, it is much more reliable to visually identify image features and make use of stereological drop-in tools, which establish the relationship between chosen features and the image as a whole and to facilitate measurement (Russ, 1995a). Drop-ins may take the form of a square array or random array of dots, a square grid of lines, a series of randomly arranged lines, a series of non-intersecting circles or a series of cycloids (Russ, 1995a). The choice of drop-in is determined by the nature of the image. The rule is that structures to be measured must be random, isotropic and uniform (Russ, 1995b). If the structure is random then successive measurements are independent and the use of an organized grid is appropriate. When structural features are isotropic they look the same from any direction and uniformity permits measurement from any location to produce the same result. Another requirement for stereological measurement is that the number of measurements must support statistical evaluation (Rhodes and Khaykin, 1986). If these basic criteria are met, structural parameters, such as volume fraction, surface area of features per unit test volume or mean free distance between image features can be determined.

A stereological approach to image quantitation provided an unbiased and efficient method to measure bubble length, lamella length (distance between the bubbles), and volume fraction of air in whipped cream. Low temperature SEM was used preserve the structure of the whipped cream. Images were digitized through Active Scan Interface (Voyager, Middleton, WI) which intercepts the X and Y signals before they reach the video boards in the SEM. However, images could just as easily be digitized later from SEM micrographs by using a scanner. The total number of pixels in the image, or P_T, represents the total number of test points in the image field. A grid with a defined pixel spacing is added to the image in the form of an overlay. This overlay should be fine enough that it does not mask important image features but large enough for easy visibility (Russ, 1995a). It is important to realize that a photographic image and a hand drawn grid overlay are equally effective for stereological measurement. Computer generated overlays can be used in imaging packages such as Adobe Photoshop as well as in conjunction with image analysis software. The points are marked, either with a pen or mouse, where the grid intersects the image feature to be quantified. The number of counts are used together with the number of grid points, the dimensions of the grid and the image area to derive stereological values.

Figure 1. Low temperature SEM image of whipped cream and grid overlay with a pixel spacing of 50 µm. Dots mark the intersection between the vertical grid lines and the bubble interface. Bar = 100 µm.

All foam images were collected at the same magnification and the dimensions of the image were calibrated using a copper grid of defined dimension. A grid was generated (Labtronics Inc. Guelph, Ontario) using a lattice spacing of 50 pixels, and overlayed on foam images through Optimas® image analysis software. Intersection points between the vertical lines of the grid and the bubbles were marked using the mouse as shown in Figure 1. The enumeration of P_H, the total number of hit points and the distance (d) between two test points leads to the determination of the total area taken up by the bubbles, (P_H)d² . This area, that is, the area fraction of the bubbles, can be calculated from A/A_T where A_T, the total test area, is determined from d²m² and m² is the total number of test points. Since A/A_T is equivalent to V/V_T (volume of feature over the total volume) (Rhodes and Khaykin, 1986) it is possible to determine volume fraction of air in the foam. The measurement of volume fraction of air bubbles in the foam will quantify the change in foam structure over time and allow for the evaluation of the effect of different treatments on foam stability.

The purpose of stereological measurement was to evaluate the effect of the choice of heat treatment and the addition of stabilizer on structural stability of whipped creams. Freshly whipped cream was compared with the foam which had been stored for 24 hours. Foam destabilization occurs through drainage of serum and also through coalescence and rise of air bubbles. Samples were taken from the top of the foam to exaggerate the effect. Changes in foam morphology after 24 h of refrigerated storage provided incite into the source of these differences. The amount of air remaining in the foam was taken as an indicator of foam stability, that is, the more stable foam held more air.

References:

1. Rhodes, M.B. and Khaykin, 1986. Foam characterization and quantitative stereology. Langmuir 2:643-649.
2. Russ, John, C., 1995a. Instructions for Using Drop-Ins for Manual Stereology, hand-out supplied by John Russ, Material Science and Engineering Department, North Carolina State University, Raleigh, NC.
3. Russ, John, C., 1995b. Computer-assisted manual stereology. Journal of Computer-Assisted Microscopy 7(1):35-46.

Book: Unbiased Stereology: Three Dimensional Measurement in Microscopy
Stereology society, courses, etc.

Polysaccharide or protein hydrocolloids are known to retard ice recrystallization in frozen systems during storage at fluctuating temperatures, but the mechanism is not clear. Hydrocolloid stabilizers were labeled with rhodamine isothiocyanate (RITC) and incorporated into solutions of sucrose (24%) and sucrose (16%) with skim milk powder (SMP) (14.7%). Solutions contained either no stabilizer or 0.3% of carrageenan, carboxymethyl cellulose (CMC), xanthan gum, sodium alginate, locust bean gum (LBG), or gelatin. For light microscopy, a small drop of the solution was placed on a glass microscope slide, cover slipped and secured within a cold stage (Linkam Scientific Instruments, UK) mounted on an Olympus BX-60 microscope. The solutions were quench frozen to -50°C, precycled in order to get similar ice crystal size at t=0 (p<0.05) and cycled between -3.5°C and -6°C, 5 times. Samples were observed using either transmitted yellow/green light or epifluorescence illumination with an Olympus rhodamine filter set. Two images per field, one transmitted and one epifluorescence, were acquired at t=0 and at -3.5°C of each cycle. Images were acquired using a Photometrics SenSys 1401E monochrome camera. Adobe PhotoShop and the Image Processing Tool Kit (1) were used for all image processing and image analysis. Quantitative image analysis was used to measure the equivalent circular diameter (2) of ice crystals in all brightfield images. Recrystallization rate was then calculated as the slope of the linear regression of the ice crystal median diameters obtained from the brightfield data.

Figure 1 shows both the brightfield and fluorescence microscopy images collected from the same field for the LBG + sucrose sample. The source of fluorescence is the labeled polysaccharide, which enables its location in the unfrozen phase to be seen. After cycling, the formation of a gel-like fluorescent structure surrounding the ice crystals was observed. Once the crystals were melted (-2°C), the LBG network remained intact.

Figure 2 shows the structures from stabilizer-sucrose solutions resulting from cycling, in the presence or absence of SMP. The only sucrose solutions in the absence of milk protein that developed a gel-like structure after cycling were those that contained locust bean gum.The recrystallization rate in these solutions was, however, similar to that in the control samples. In contrast, the recrystallization rate was significantly reduced by alginate and xanthan (p>0.05) (Table 1). Conversely, gelatin was the only stabilizer tested which did not retard ice recrystallization in sucrose-skim milk solutions (Table 1). It was observed to form distinctive gels with milk proteins. Similar gels were also formed in the presence of locust bean gum or carrageenan in sucrose solutions which contained milk solids (Figure 2).

It is therefore suggested that steric blocking of the interface or inhibition of solute transport to and from the ice interface caused by the gelation of the polymer, is not the only mechanism of stabilizer action. However, a structural molecular arrangement that keeps the melting water in close proximity to the ice crystal surface and ensures that this water refreezes onto the original crystal rather than migrating to the surface of a larger crystal elsewhere, must be present. The molecular interactions between polysaccharides and proteins appear to be key factors in retarding ice recrystallization.

References

(1) Russ, C. and Russ, J. C., Impage Processing ToolKit, http:// www.reindeergraphics.com
(2) Russ, J. C. 1998. In "The Image Processing Handbook" 3rd edition, CRC Press, Boca Raton, FL, USA

Updated: May 6, 2006.

FOODS UNDER THE MICROSCOPE