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

Abstract

Background: 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 Ca(2+), 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.

Results: 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.

Conclusion: 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.

Keywords: Cilium length; Measurements; Methods; Primary cilia.

Fig. 1

Visual appearance of a primary cilium. a. Scanning Electron Microscopy image in the developing heart showed a continuous slope of the endothelial primary cilium. Scale bar is 1 μm. Re-used with permission and adapted from van der Heiden et al., 2006 [33]. b Schematic overview of the difference between real length of the cilium and visibility on a maximum intensity projection

Fig. 2

Visual appearance of the same cilium with the different methods. Visual appearance of the same angled cilium with the MIP method (a), PyT method (b) (see also Additional file 4: Movie S2), DAAS method (c), and a reconstruction with Amira (d). Scale bars are 3 μm. e. Length of a cilium, measured with different sizes of the z-slices using the PyT method. Dotted line indicates z-slice size of 0.25 µm, and dashed line indicates the cilium length (2.26 μm) measured by 3D reconstruction using Amira

Fig. 3

The PyT and the DAAS methods. a. PyT method. With the assumption of a continuous slope, the Pythagorean theorem can be applied to calculate the length of a cilium. b–d. DAAS method. After selecting a cilium and the cross section line (b), the program gives a top view (c) and the vertical slice of the selected plane (d). Measurements are performed in Matlab. Cilia are measured on grayscales from 55 to 255

Fig. 4

Cilium length between the different populations measured in three different methods. a Measurements with the MIP method. b Measurements with the PyT method. c Measurements with the DAAS method. In all methods, the same sample of cilia is used. Boxplot contains the 25–75 percentile, and the whiskers represent the minimum and maximum of the measurements. n = 31 for both the flat and angled cilia

Fig. 5

Comparison of the flat and angled cilia. a Comparing the different methods for the population of flat cilia. b Comparing the different methods for the population of angled cilia. ICC = intraclass correlation coefficient. Interpretation of the ICC according to Landis and Koch, 1977: kappa value of <0.2 = slight agreement; >0.2–0.4 = fair; >0.4–0.6 = moderate; >0.6–0.8 = substantial; >0.8 = almost perfect agreement. n = 31 for both the flat and angled cilia