We describe a technique for simultaneous quantification of the contractile forces and cytosolic calcium dynamics of muscle fibers embedded in three-dimensional biopolymer gels under auxotonic loading conditions. We derive a scaling law for linear elastic matrices such as basement membrane extract hydrogels (Matrigel) that allows us to measure contractile force from the shape of the relaxed and contracted muscle cell and the Young's modulus of the matrix without further knowledge of the matrix deformations surrounding the cell and without performing computationally intensive inverse force reconstruction algorithms. We apply our method to isolated mouse flexor digitorum brevis (FDB) fibers that are embedded in 10 mg/mL Matrigel. Upon electrical stimulation, individual FDB fibers show twitch forces of 0.37 ± 0.15 μN and tetanic forces (100-Hz stimulation frequency) of 2.38 ± 0.71 μN, corresponding to a tension of 0.44 ± 0.25 kPa and 2.53 ± 1.17 kPa, respectively. Contractile forces of FDB fibers increase in response to caffeine and the troponin-calcium stabilizer tirasemtiv, similar to responses measured in whole muscle. From simultaneous high-speed measurements of cell length changes and cytosolic calcium concentration using confocal line scanning at a frequency of 2048 Hz, we show that twitch and tetanic force responses to electric pulses follow the low-pass filtered calcium signal. In summary, we present a technically simple high-speed method for measuring contractile forces and cytosolic calcium dynamics of single muscle fibers. We expect that our method will help to reduce preparation time, costs, and the number of sacrificed animals needed for experiments such as drug testing.

中文翻译：

---

高速牵引显微镜测量骨骼肌纤维收缩力

我们描述了一种同时量化肌肉纤维的收缩力和细胞溶质钙动力学的技术，该技术嵌入在 3D 生物聚合物凝胶中，在 auxotonic 加载条件下。我们推导出线性弹性矩阵（例如基底膜提取物水凝胶（Matrigel））的标度定律，使我们能够从松弛和收缩的肌肉细胞的形状和矩阵的杨氏模量中测量收缩力，而无需进一步了解周围的矩阵变形细胞并且不执行计算密集的反力重建算法。我们将我们的方法应用于嵌入 10 mg/mL Matrigel 的分离的小鼠屈肌短屈肌 (FDB) 纤维。电刺激后，单个 FDB 纤维显示 0.37 ± 0 的抽搐力。15 μN 和强直力（100 Hz 刺激频率）为 2.38 ± 0.71 μN，分别对应于 0.44 ± 0.25 kPa 和 2.53 ± 1.17 kPa 的张力。FDB 纤维的收缩力响应咖啡因和肌钙蛋白-钙稳定剂 tirasemtiv 增加，类似于在整个肌肉中测量的反应。从使用 2048 Hz 频率的共焦线扫描同时高速测量细胞长度变化和细胞溶质钙浓度，我们表明对电脉冲的抽搐和强直力响应遵循低通滤波钙信号。总之，我们提出了一种技术上简单的高速方法，用于测量单个肌肉纤维的收缩力和细胞溶质钙动力学。我们希望我们的方法将有助于减少准备时间、成本、