Foods Under the Microscope - Ice Cream Structure by Freeze-Substitution

Thin-Section Transmission Electron Microscopy of Ice Cream after Freeze Substitution

by H. Douglas Goff, Ph.D.

This narrative of our visualization of ice cream structure by freeze-substitution (cryo-fixation) transmission electron microscopy (TEM) was prepared for Dr. Miloslav Kalab, Foods Under the Microscope". It is my second contribution for Dr. Kalab. The first can be seen here.
 
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.
 
cross sections of ice cream at low magnification
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: 817-829.