Table 2.

CiliaQ settings for presented datasets

	Figure 1b, c, d, f, g: mIMCD-3 cells	Figure 3: MEFs	Fig. 4: IMCD-3	Figures 5, 6 and 7: Zebrafish embryos
Microscopy and input image specifications
Microscope	Nikon Eclipse Ti	Nikon Eclipse Ti	Nikon Eclipse Ti	Zeiss Examiner Z1
Channel 1	DAPI	DAPI	DAPI	DAPI
Channel 2	ARL13B antibody	SMO antibody	acTUB antibody	acTUB antibody
Channel 3	gTUB antibody	ARL13B antibody	ARL13B antibody	ARL13B-GFP
Objective	Plan-Apochromat 60x oil immersion	Plan-Apochromat 60x oil immersion	Plan-Apochromat $60\times$ oil immersion	Plan-Apochromat $20\times$/0.8 M27
Calibration (voxel width) ($\mathrm{\mu }\text{m}$)	0.1035801	0.2067050	0.1035801	0.1383776
Voxel depth ($\mathrm{\mu }\text{m}$)	0.5	0.5	0.5	1.0
CiliaQ Preparator
Channel to be segmented (i.e., reconstruction channel)
Channel Nr	2	3	2	2
Include unsegmented copy	Yes	Yes	Yes	No
Subtract background before segmentation	No	No	No	No
Subtract background: radius
Divide by background before segmentation	No	No	No	Yes
Divide by background: radius				$1.5\mathrm{\mu }\text{m}$
Smooth with Gaussian	No	No	No	Spinal cord: No; Tel.: Yes
Smooth with Gaussian: sigma				Tel.: 0.25
Segmentation method	Renyi Entropy or Canny3D (CiliaQ Preparator run twice, both output images analyzed)	Triangle	Otsu	Hysteresis threshold (custom)
				Low threshold:
				$\bullet$ Spinal cord:1.8
				$\bullet$ Tel.: 1.85
				High threshold:
				$\bullet$ Spinal cord: 2.8
				$\bullet$ Tel.: 3.0
Stack handling	Apply threshold determined in a maximum intensity projection	Apply threshold determined in a maximum intensity projection	Apply threshold determined in a maximum intensity projection
More channels to be segmented
Segment a second channel	No	No	Yes	No
Channel Nr			3
Include unsegmented copy			Yes
Subtract Background Before Segmentation			No
Subtract Background: radius
Segmentation method			Triangle
Stack handling			Apply threshold determined in a maximum intensity projection
CiliaQ
Detection
Measure intensity A	No	Yes	Yes	Yes
Measure intensity B	No	No	Yes	No
Basal stain present	Yes	No	No	No
Channels: reconstruction	2	3	2 or 4 (CiliaQ run twice)	2
Channels: opt. intensity A		2	5 or 3	3
Channels: opt. intensity B			3 or 5
Channels: basal stain	4
Minimum cilium size (voxel)	10	10	10	10
Increase range for connecting cilia	No	No	No	No
Additionally exclude...	Nothing	Cilia touching x or y or z borders	Cilia touching x or y or z borders	Cilia touching x or y or z borders
Minimum size of intensity regions...(voxel)	1	1	1	1
Increase range for connecting intensity regions	No	No	No	No
Determine skeleton-based results	Yes	Yes	Yes	Yes
Before skeletonization: Gauss filter XY sigma	1.0 (for Fig. 1f and g CiliaQ was run multiple times with different sigmas between 0.0 and 5.0)	1.0	1. 0	1.0
Before skeletonization: gauss filter Z sigma	0.0	0.0	0.0	0.0

Settings not applicable for the analysis result (e.g., visualization settings, settings left to default parameters) were not included in this table. Empty cells indicate that the entered value in the settings dialog is obsolete. Abbreviations: mIMCD-3 (mouse inner medullary collecting duct 3), MEFs (mouse embryonic fibroblasts), acTUB (acetylated Tubulin), gTUB (gamma Tubulin), tel (Telencephalon), voxel (represents a pixel in a 3D image)