Cilia Research Laboratory, College of Public Health Medical and Veterinary Sciences, James Cook University, Townsville, Queensland, Australia.
Biomedical Imaging Facility, University of New South Wales, Sydney, New South Wales, Australia.
Physiol Rep. 2022 Jun;10(11):e15349. doi: 10.14814/phy2.15349.
Motile cilia are found in numerous locations throughout our body and play a critical role in various physiological processes. The most commonly used method to assess cilia motility is to quantify cilia beat frequency (CBF) via video microscopy. However, a large heterogeneity exists within published literature regarding the framerate used to image cilia motility for calculating CBF. The aim of this study was to determine the optimal frame rate required to image cilia motility for CBF assessment, and if the Nyquist theorem may be used to set this rate. One-second movies of cilia were collected at >600 fps from mouse airways and ependyma at room-temperature or 37°C. Movies were then down-sampled to 30-300 fps. CBF was quantified for identical cilia at different framerates by either manual counting or automated MATLAB script. Airway CBF was significantly impaired in 30 fps movies, while ependymal CBF was significantly impaired in both 60 and 30 fps movies. Pairwise comparison showed that video framerate should be at least 150 fps to accurately measure CBF, with minimal improvement in CBF accuracy in movies >150 fps. The automated script was also found to be less accurate for measuring CBF in lower fps movies than manual counting, however, this difference disappeared in higher framerate movies (>150 fps). In conclusion, our data suggest the Nyquist theorem is unreliable for setting sampling rate for CBF measurement. Instead, sampling rate should be 3-4 times faster than CBF for accurate CBF assessment. Especially if CBF calculation is to be automated.
纤毛存在于我们身体的许多部位，在各种生理过程中起着关键作用。评估纤毛运动最常用的方法是通过视频显微镜来量化纤毛拍频（CBF）。然而，在有关用于计算 CBF 的纤毛运动成像的帧率方面，发表的文献中存在很大的异质性。本研究旨在确定用于 CBF 评估的纤毛运动成像所需的最佳帧率，以及是否可以使用奈奎斯特定理来设置该帧率。在室温或 37°C 下，从小鼠气道和室管膜中以>600fps 的速度采集纤毛 1 秒的电影。然后将电影下采样到 30-300fps。通过手动计数或自动 MATLAB 脚本，在不同帧率下对相同的纤毛进行 CBF 定量。在 30fps 的电影中，气道 CBF 显著受损，而在 60 和 30fps 的电影中，室管膜 CBF 均显著受损。成对比较表明，视频帧率至少应为 150fps 才能准确测量 CBF，帧率>150fps 时 CBF 准确性的提高最小。还发现，与手动计数相比，自动脚本在较低帧率的电影中测量 CBF 的准确性较低，但在较高帧率的电影（>150fps）中，这种差异消失。总之，我们的数据表明奈奎斯特定理不可靠用于设置 CBF 测量的采样率。相反，采样率应该是 CBF 的 3-4 倍，以进行准确的 CBF 评估。特别是如果要自动计算 CBF。
