The Application of cryo-SEM for the Study of the Microstructure of

							 The Application of cryo-SEM for the Study of the Microstructure of
                            Milk Gels

                       Lavochkin M.1, Zuckerman H.1 and Schmidt J.2
      1
          Department of Food Engineering & Biotechnology, Technion, Haifa, Israel
               2
                 Department of Chemical Engineering, Technion, Haifa, Israel

Understanding the food microstructure is of great importance for process and product
development having great effects on the sensory characteristics. Since biological and
food materials contain high amounts of water, they can not be observed using a
Scanning Electron Microscope (SEM) without removing the water. However, cryo-
SEM can be used to study the microstructure of hydrated samples. The goal of the low-
temperature cryo-SEM is to vitrify the liquid phase with all the constituents, i.e.
macromolecules, thus preserving them in their natural and original state.

The initial rapid cooling of the sample is the most critical part in the use of the cryo-
SEM system. A slush of LN2 (-2100C) is used for the fast freezing to minimize any
damage ice crystals may cause. After freezing, the specimen is placed onto a cold stage
where it is being fractured exposing the internal surface of sample. Etching of the
sample is the next process, in which water is sublimed from the surface of the sample
exposing the underlying structural features. Sublimation of ice under the microscope
vacuum starts at –1000C.

The purpose of this study was to optimize the etching time and temperature of milk
gels, which are a protein matrix (casein), causing minimum ice artifacts from secondary
nucleation. Secondary nucleation may occur during the temperature rise used for
etching, which can damage the specimen. Optimization of etching temperature and time
is needed for the study of the milk gel microstructure in the cryo-SEM.

The milk gels were prepared from 1-% skim milk by adding the enzyme rennet at pH
6.0. The samples were incubated for 1.5 h at 350C, allowing gelation, and were then
stored at 4 0C for ~12h. Structure observations were preformed using the CT1500 –cryo
system (Gatan LTd., UK) mounted on a Scanning Electron Microscope (JSM-5400,
JEOL). Samples were etched at –900C or –950C for predetermined times followed by
gold coating. All samples were observed at –1500C under 15 kV.

The structure of the milk gels at the various combination of etching times and
temperatures are presented in the Figures below.
                                              A                                                          B
Figure1: Milk gel etched at –900C. (A) 7.5 min, (B) 12.5min.




                                              A                                                          B

Figure 2: Milk gel etched at –950C. (A) 12.5 min, (B) 17.5 min.


We recommend that, the –900C and 12.5 min of etching will be used for further
microstructure studies of milk gels.
References
1. Goldstein, J. I., Roming, A. D., Newbury, D. E., Lyman, C. E., Echlin, P., Fiori C., Joy, D. C. and
Lifshin, E. 1992. Scanning electron microscopy and X-ray microanalysis. Plenum press New York and
London. pp. 644-651.
2. Echlin P. 1992. Low-temperature microscopy and analysis .Plenum press. pp. 141-191, 349-411.