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Measuring the primary cilium length: improved method for unbiased high-throughput analysis

机译：测量初级纤毛长度：用于无偏高通量分析的改进方法

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Primary cilia are cellular protrusions involved in mechanic and chemical sensing on almost all cells of our body. Important signaling pathways, including Hedgehog, TGFβ, and Ca2+, are linked to cilia and/or cilia function. Cilia can vary in length, which has functional implications. To measure these lengths correctly, a standardized method with high reliability and throughput is required. To date, methods for length measurements in cultured cells after fluorescent staining for ciliary components are error prone with a possible human selection bias, primarily caused by the orientation of cilia with respect of the imaging plane. In tissue sections, accurate measurements become an even larger challenge due to additional random sectioning plane. Cilia can be reconstructed in 3D and measured one by one, but this is a labor-intensive procedure. Therefore, we developed a new, high-throughput method with less selection bias. To identify the optimal type of measurement of straight and relatively short cilia, three methods were compared. The first method is based on maximum intensity projection (MIP), the second method is based on the Pythagorean theorem (PyT), and the third is based on 3D alternative angled slicing (DAAS). We investigated whether cilia visible in the plane of focus (‘flat cilia’), and the ones that are angled with respect to the plane of focus are represented differently among the various methods. To test the agreement between the methods, intraclass correlations are calculated. To measure flat cilia, MIP and DAAS provided representative results, with the MIP method allowing for higher throughput. However, when measuring the angled cilia with MIP, the actual cilium length is overtly underestimated. DAAS and PyT are exchangeable methods for length measurements of the angled cilia, while PyT exhibits higher throughput and is therefore the preferred method for measuring the length of an angled cilium. PyT is a universal measuring method to measure straight cilia, without selection bias. MIP provides similar results for flat cilia, but underestimates the length of angled cilia. In addition, PyT facilitates high-throughput length measurements. Manual tracking or reconstruction will be the method of choice to measure irregularly shaped cilia.
机译：原发性纤毛是涉及人体几乎所有细胞的机械和化学感应的细胞突起。重要的信号传导途径，包括刺猬，TGFβ和Ca2 +，与纤毛和/或纤毛功能相关。纤毛的长度可以变化，这具有功能上的含义。为了正确地测量这些长度，需要具有高可靠性和吞吐量的标准化方法。迄今为止，在对纤毛成分进行荧光染色后，在培养的细胞中进行长度测量的方法容易出错，可能存在人类选择偏倚，这主要是由于纤毛相对于成像平面的方向引起的。在组织切片中，由于需要额外的随机切片平面，因此精确的测量成为更大的挑战。纤毛可以用3D重建并一个接一个地进行测量，但这是一项劳动密集型的过程。因此，我们开发了一种新的高通量方法，具有较小的选择偏差。为了确定直纤毛和相对短纤毛的最佳测量类型，比较了三种方法。第一种方法基于最大强度投影（MIP），第二种方法基于毕达哥拉斯定理（PyT），第三种方法基于3D替代角度切片（DAAS）。我们研究了在聚焦平面中可见的纤毛（“平坦纤毛”）以及相对于聚焦平面成角度的纤毛在各种方法中的表示方式是否不同。为了测试方法之间的一致性，计算类内相关性。为了测量扁平纤毛，MIP和DAAS提供了代表性的结果，而MIP方法可实现更高的通量。但是，用MIP测量成角度的纤毛时，实际纤毛长度被低估了。 DAAS和PyT是用于测量角纤毛长度的可交换方法，而PyT表现出更高的通量，因此是测量角纤毛长度的首选方法。 PyT是一种测量直纤毛的通用测量方法，没有选择偏倚。 MIP可为扁平纤毛提供相似的结果，但会低估成角度的纤毛的长度。此外，PyT有助于进行高通量长度测量。手动跟踪或重建将是测量不规则形状的纤毛的选择方法。

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