We investigated the applicability of cryo-electron tomography as a method to quantify changes in the major constituents of casein micelles (i.e., casein proteins, putative colloidal calcium phosphate nanoclusters, and serum-filled voids and channels) in response to their environment. Skim milk diluted 20-fold in milk serum was used for this study. Tomograms were generated for multiple casein micelles at 2 different pH values (6.7 and 6.0) and pixel intensity thresholds were identified for each constituent. The volume of each constituent was determined using these thresholds and expressed as a fraction of micelle volume. At the given dilution, a significant decrease in the volume fractions of casein proteins (∼37%) and putative colloidal calcium phosphate nanoclusters (∼67%) was observed with the reduction of pH from 6.7 to 6.0. Assessment of casein micelle fraction obtained by ultracentrifugation of corresponding skim milk samples produced comparable results. When using such an approach, the imaging conditions, denoising methods, and thresholding approaches used can all affect the precision of the measurements, but the overall trends in constituent volumes are able to be tracked. The primary advantage of using cryo-electron tomography is that analysis can be done at the level of individual micelles, within a 3-dimensional morphological context. This workflow paves the way for high-throughput exploration of milk micelles and how their environment shapes their composition and structure.

中文翻译：

---

使用低温电子层析成像技术评估牛酪蛋白胶束的组成体积和形态。

我们调查了低温电子断层扫描作为量化酪蛋白胶束主要成分（即酪蛋白，假定的胶体磷酸钙纳米簇以及充满血清的空隙和通道）的变化的方法的适用性。本研究使用在乳浆中稀释20倍的脱脂乳。在2个不同的pH值（6.7和6.0）下针对多个酪蛋白胶束生成了断层图，并为每种成分确定了像素强度阈值。使用这些阈值确定每种成分的体积，并表示为胶束体积的一部分。在给定的稀释度下，随着pH从6.7降低到6.0，酪蛋白蛋白（〜37％）和胶体磷酸钙纳米簇（〜67％）的体积分数显着降低。通过对相应脱脂奶样品进行超速离心获得的酪蛋白胶束级分的评估产生了可比的结果。当使用这种方法时，所使用的成像条件，去噪方法和阈值处理方法都可以影响测量的精度，但是可以跟踪组成量的总体趋势。使用低温电子断层扫描的主要优点是可以在3维形态学背景下在单个胶束的水平上进行分析。该工作流程为牛奶胶束的高通量探索及其环境如何影响其组成和结构铺平了道路。使用的阈值方法都会影响测量的精度，但是可以跟踪组成量的总体趋势。使用低温电子断层扫描的主要优点是可以在3维形态学背景下在单个胶束的水平上进行分析。该工作流程为牛奶胶束的高通量探索及其环境如何影响其组成和结构铺平了道路。使用的阈值方法都会影响测量的精度，但是可以跟踪组成量的总体趋势。使用低温电子断层扫描的主要优点是可以在3维形态学背景下在单个胶束的水平上进行分析。该工作流程为牛奶胶束的高通量探索及其环境如何影响其组成和结构铺平了道路。