Error correction of confocal microscopy images for in situ food microstructure evaluation

Image processing methods were developed for correcting confocal microscopy images for errors introduced during image acquisition and subsequent three-dimensional (3-D) image reconstruction. These include corrections for aberrations due to refractive index mismatch, light attenuation with sample depth, uneven intensity across image layers, and image misalignment. These procedures were validated using a simulated image with different illumination conditions. As example applications, dynamic changes in microstructure of process cheese during heating and gelation of β-lactoglobulin (whey protein) were studied in situ. The error-corrected image layers were used to reconstruct a 3-D composite image of fat globules in cheese. The techniques presented will allow objective evaluation of in situ changes in 3-D microstructural features in different foods.