Traction force microscopy has been established as the accepted method for evaluating cell-induced mechanical stresses to their microenvironments, typically using two-dimensional (2D) elastic, synthetic gel-substrates. As cells naturally experience 3D environments in vivo, traction microscopy has been adapted to 3D gels; cells can be tracked over time in 3D. Microscopy images acquired in several fields-of-view e.g. in a time series, may experience drift, which can produce artefactual results that may appear valid and lead to flawed analysis. Hence, we have developed an algorithm for 2D/3D de-drifting of cell-images on 3D gels with fiducial markers (beads) as anchor points. Both lateral and vertical de-drifting are performed using gel-internalized beads, as those used in traction microscopy experiments; this eliminates need for immobilizing beads under the gel for de-drifting, and reduces experiment time. We introduce simulations of initially grid-ordered dots (beads) that are radially displaced to experimentally observed distances, while also applying additive drift. This facilitates testing and demonstration of the de-drifting procedures in 2D/3D. We demonstrate the importance of applying de-drifting using both computer-simulated drifts and experimentally observed drifts in confocal microscopy images. We show that our de-drifting algorithm can remove lateral and/or vertical drift revealing even small, underlying signals. The 2D/3D de-drifting algorithm, crucial for accurate identification of cell-induced marker-displacement, as well as the bead simulations, will shorten traction microscopy experiments and facilitate optimization of the experimental protocols.

牵引力显微镜已被确立为评估细胞诱导的对其微环境的机械应力的公认方法，通常使用二维（2D）弹性，合成凝胶基质。随着细胞自然地在体内体验3D环境，牵引显微镜已适应3D凝胶；可以随时间推移以3D方式跟踪单元。在多个视场（例如，一个时间序列）中采集的显微图像可能会发生漂移，从而产生的伪像结果可能看起来有效，并导致分析结果有误。因此，我们开发了一种使用基准标记（珠子）作为锚点的3D凝胶上细胞图像的2D / 3D反漂移算法。使用凝胶内在的珠子进行横向和垂直的去漂移，就像在牵引显微镜实验中使用的那样。这消除了将珠子固定在凝胶下进行脱水的需要，并减少了实验时间。我们介绍了对最初按网格顺序排列的点（珠子）进行模拟的方法，这些点沿径向方向移动到实验观察到的距离，同时还应用了附加漂移。这有助于在2D / 3D中测试和演示去漂移过程。我们证明了使用共聚焦显微镜图像中的计算机模拟漂移和实验观察到的漂移应用去漂移的重要性。我们表明，我们的去漂移算法可以消除横向和/或垂直漂移，从而揭示甚至很小的潜在信号。2D / 3D去漂移算法对于精确识别细胞诱导的标记位移以及微珠模拟至关重要，它将缩短牵引显微镜实验并促进实验方案的优化。我们证明了我们的去漂移算法可以消除横向和/或垂直漂移，从而揭示甚至很小的潜在信号。2D / 3D去漂移算法对于精确识别细胞诱导的标记位移以及微珠模拟至关重要，它将缩短牵引显微镜实验并促进实验方案的优化。我们证明了我们的去漂移算法可以消除横向和/或垂直漂移，从而揭示甚至很小的潜在信号。2D / 3D去漂移算法对于精确识别细胞诱导的标记位移以及微珠模拟至关重要，它将缩短牵引显微镜实验并促进实验方案的优化。