Quantitative analysis of protein gel structure by confocal laser scanning microscopy

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S. Richter, A. Schwedt (Eds.): EMC 2008, Vol. 2: Materials Science, pp. 757–758, DOI: 10.1007/978-3-540-85226-1_379, © Springer-Verlag Berlin Heidelberg 2008

Quantitative analysis of protein gel structure by confocal laser scanning microscopy

Komla Ako, Lydiane Bécu, Taco Nicolai, Jean-Cristophe Gimel, Dominique Durand Polymères, Colloïdes, Interfaces, UMR CNRS, Université du Maine, 72085 Le Mans

Cedex 9, France [email protected]

Keywords: Quantitative, gel structure, confocal

Scattering techniques are suited to quantitatively study the structure of protein gels [1, 2] at length-scales smaller than one micron, but it is clear from microscopy images that protein gels may exhibit structural features at larger length scales [3]. Microscopy has been used for qualitative comparisons of protein gels formed at different conditions, but little has been done to extract quantitative information comparable to that yielded by scattering methods.

We have developed a novel approach to study the microscopic structure of protein gels and aggregates by quantitative analysis of images obtained by confocal laser scanning microscopy (CLSM). The pair correlation function and the structure factor of the images were calculated numerically "Figure 1." We have established that if proper care is taken the pixel intensities of the images are proportional to the protein concentration. Therefore the result of this analysis is equivalent to scattering measurements, but on larger length scales "Figure 2." We will show that for isotropic systems, such as protein gels, the analysis of 2-D and 3-D images give identical results, but the spatial resolution of the former is better.

We used confocal microscopy to quantify the effect of electrostatic interaction on the structure of protein gels by varying the pH and the ionic strength. This study extends a recent investigation using scattering techniques [1, 4, 5] to more heterogeneous gels formed closer to the isoelectric point or in the presence of more salt. We also studied mixed protein/polysaccharide gels for which the structure is determined by the competition between aggregation and phase separation [6, 7].

1. Durand, D.; Gimel, J.C.; Nicolai, T. (2002), Physica A, 304, 253.

2. Nicolai, T.; Durand, D. (2007) Curr. Opin. Colloid Interface Sci., 12, 23 3. Olsson, C.; Langton, M.; Hermansson, A.-M. (2002) Food Hydrocolloids, 16, 111

4. Nicolai T. In Food Colloids, Self-Assembly and Material Science; Dickinson, E.; Leser, M.

E., Eds.; RSC Publishing: Cambridge, 2007; pp 35-56.

5. Mehalebi, S.; Nicolai, T.; Durand, D. to be published

6. Baussay, K.; Nicolai, T.; Durand, D. (2006) Biomacromolecules, 7, 304.

7. Baussay, K.; Nicolai, T.; Durand, D. (2006) J. Coll. Int. Sci, 304, 335

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Figure 1. (Left) Semi-logarithmic representation of the pair correlation functions of heat-set β-lg gels formed at pH 7 and different NaCl concentrations. The solid lines represent fits to

g ( r ) = B

₁

exp[ − ( r / ξ )

^β

] + 1

. (Right) Double logarithmic representation of the master curve of the data shown at the left hand obtained by normalizing (g(r)-1)with the amplitude and r with the correlation length. The straight line gives an approximate description of the data over a limited range of r and could mistakenly be interpreted as a self similar structure. The inset shows (g(r) -1)/B1.

C_s (M)

0.01 0.1 1

ξ (nm)

10¹ 10² 10³

Figure 2. Dependence of the correlation length of heat-set β-lg gels formed at pH 7 on the NaCl concentration. Circles represent data obtained with light scattering and squares represent data obtained with CSLM.

1 r/ξ 10

(g(r)-1)/B1

-0.1 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0

1 r/ξ (g(r)-1)/B1

0.01 0.1 1

r(µm)

1 10

g(r)

0.9 1.0 1.1 1.2 1.3 1.4 1.5 1.6 1.7 1.8 1.9 2.0

2.1 0.15M

0.20M 0.25M 0.28M 0.6M 0.8M 1.0M 2.0M