Table 1.

Summary of commonly used software including costs and basic image processing features.

Software name	Developer/parent company	# of papers	Cost	Thresholding methods	Segmentation methods	Quantified cell features
ImageJ	National Institutes of Health	56	Free	Options: Global, Local, Auto (global histogram-derived) Threshold. Auto Options: Huang, Intermodesl, IsoData, Li, MaxEntropy, Mean, MinError(l), Minimum, Moments, Otsu, Percentile, RenyiEntropy, Shanbhag, Triangle, Yen (ImageJ Docs Release 1.17.2)	Options: Multiple Methods: Trainable Weka Segmentation, Auto Thresholding, and Masking, Manual Segmentation (ImageJ Docs Release 1.17.2)	Area (Debertin et al., 2015; Hannibal et al., 2017), Cell Density (Virgone-Carlotta et al., 2013; Jawaid et al., 2018), Count (Huang et al., 2012; Li et al., 2013; Ferber and Tiram, 2014; Bendali et al., 2015; Debertin et al., 2015; Sakita et al., 2016; Seidl and Rubel, 2016; Van Der Woude and Smid, 2016; Žygelyte et al., 2016; Banerjee and Chaturvedi, 2017; Hannibal et al., 2017; Wang et al., 2017; Jawaid et al., 2018; Santos et al., 2018), Colocalization (Alcami and Marty, 2013; Li et al., 2013; Virgone-Carlotta et al., 2013; Banerjee and Chaturvedi, 2017; Sousa-Valente et al., 2017), Dendritic Diameter (Jawaid et al., 2018), Diameter (Sakita et al., 2016; Seidl and Rubel, 2016; Hannibal et al., 2017), Feret's Diameter (Herron and Miles, 2012), Intensity (Dibaj et al., 2010; Fernández-Alvarez et al., 2011; Masuda et al., 2011; Smith et al., 2011; Hovis et al., 2012; Tapia et al., 2012; Trouche et al., 2013; Sousa-Valente et al., 2017; Awadová et al., 2018; Chu et al., 2020), Length (Seidl and Rubel, 2016; Van Der Woude and Smid, 2016), Location (Chu et al., 2020), Nuclei Count (Trouche et al., 2013), Perimeter (Sakita et al., 2016), Process (branch/dendrite) Count (Masuda et al., 2011; Santos et al., 2018), Process (branch/dendrite) Length (Masuda et al., 2011; Awadová et al., 2018; Santos et al., 2018), Process Extension (Masuda et al., 2011), Puncta Size (Alcami and Marty, 2013), Puncta Intensity (Alcami and Marty, 2013), Puncta Circularity (Alcami and Marty, 2013), Thickness of Processes (branches/dendrites) (Sakita et al., 2016; Tavares et al., 2017), Total Length of Processes (branches/dendrites) (Tavares et al., 2017), Sholl Analysis (Tavares et al., 2017; Santos et al., 2018), Spheroid quantification (Martin et al., 2013), Surface Area (Cain et al., 2011; Nagel et al., 2012; Azaripour et al., 2018), Volume (Goldsmith et al., 2010; Cain et al., 2011; Heinze et al., 2013; Van Der Woude and Smid, 2016; Awadová et al., 2018)
Imaris	Oxford Instruments	23	$13,000–$45,000*	Options: threshold cutoff, baseline subtraction, background subtraction, connective baseline (Imaris Reference Manual V 5.5.0)	Options: FilamentTracer or Segmentation “Wizards: (1) AutoPaths (no loops) for a tree-like filament or Threshold (loops). (Imaris Reference Manual V 5.5.0)	Area (Bayerl et al., 2016), Branch count (Althammer et al., 2020), Branch Ends (Huang et al., 2021), Branch Order (Huang et al., 2021), Cell Distribution Profiles Across Regions (Paul et al., 2014), Colocalization (Pihlaja et al., 2008; Sosa et al., 2013; Testen et al., 2020), Count (Choi et al., 2010; Sosa et al., 2013; Bendali et al., 2015; Bayerl et al., 2016; Miller and Rothstein, 2016; Huang et al., 2021), Density (Bayerl et al., 2016), Distance between Cells (Huang et al., 2021), Filament Length (Althammer et al., 2020), Intensity (Bayerl et al., 2016), Intersections (Dando et al., 2019), Length (Choi et al., 2010; Huang et al., 2021), Location (Choi et al., 2010), Sholl Analysis (Althammer et al., 2020), Surface Area (Althammer et al., 2020), Volume (Azaripour et al., 2018; Althammer et al., 2020)
MATLAB	Mathworks	22	$50 base	Coding language	Coding language	Coding language
Amira	Thermo Scientific	20	$4,000 + (Estimate)	Options: auto-thresholding or multi-thresholding. specific algorithms are not stated. (users guide to Amira software 2019)	Options: multi-thresholding (users guide to Amira software 2019)	Area (Williams et al., 2010), Cell coverage (Williams et al., 2010), Colocalization (Virgone-Carlotta et al., 2013), Count (Choi et al., 2010; Kelber et al., 2010; Elliott et al., 2015), Density (Virgone-Carlotta et al., 2013), Distance between cells (Williams et al., 2010), Length (Choi et al., 2010; Kelber et al., 2010; Virgone-Carlotta et al., 2013), Process (Branch/Dendrite) Count (Elliott et al., 2015), Process (Branch/Dendrite) intersections (Elliott et al., 2015), Sholl Radius (Virgone-Carlotta et al., 2013), Volume (Kelber et al., 2010; Heinze et al., 2013)
Adobe Photoshop	Adobe Inc.	15	$240/year	Options: A threshold filter option based on a user input single value (Adobe Photoshop Elements User Guide Updated in 2021).	Options: segmentation is possible with a user-created workflow (Adobe Photoshop Elements User Guide Updated in 2021)	Image adjustments were made with Adobe Photoshop, but no features were quantified.
Zeiss ZEN	Zeiss	8	$2,611 per license with basic modules	Options: manual, histogram based, size, shape, intensity, and ROI based (Zen Blue Image Analysis Guide 2016).	Options: Thresholding-Based (Zen Blue Image Analysis Guide 2016)	Colocalization (Sousa-Valente et al., 2017), Intensity (Sousa-Valente et al., 2017)
MetaMorph	Molecular Devices	7	$6,000 + base price, $750 + yearly upgrades, $1,200 offline analysis cost	Options: thresholding available in region of interest selection, Authothreshold, watershed (Fundamentals of MemaMorph Workshop Slides)	Options: watershed segmentation (Fundamentals of MemaMorph Workshop Slides)	Area coverage (Williams et al., 2010; Naguib et al., 2012), Colocalization (Li et al., 2013), Count (Choi et al., 2010; Li et al., 2013; Bolea et al., 2014), Distance between cells (Williams et al., 2010), Intensity (Ghiretti and Paradis, 2011; Naguib et al., 2012), Length (Choi et al., 2010), Synapse Density (Ghiretti and Paradis, 2011)
Volocity	PerkinElmer	7	$6,230 base	Options: manual threshold, automatic threshold (OTSU method). (Volocity User Guide V 22.0)	Volocity user guide does not include the term “segment” or “segmentation.” (Volocity User Guide V 22.0)	Area (Bayerl et al., 2016), Axon length (Kemp et al., 2016), Cell density (Bayerl et al., 2016), Colocalization (Ugbode et al., 2014; Wang et al., 2017), Count (Bayerl et al., 2016; Kemp et al., 2016), Intensity (Bayerl et al., 2016; Chu et al., 2020), Size (Kemp et al., 2016), Surface Area (Bagheri et al., 2015), Volume (Bagheri et al., 2015; Bayerl et al., 2016; Chu et al., 2020)
Image-Pro Plus	Media Cybernetics	4	$5,775	Options: manual threshold, automatic threshold (OTSU method). (Image-Pro Plus Version 7.0)	Options: manual threshold based segmentation and watershed algorithm. (Image-Pro Plus Version 7.0)	Axon length (Kemp et al., 2016), Colocalization (Kemp et al., 2016), Count (Kemp et al., 2016), Vessel diameter (Tan et al., 2013)
Avizo	Thermo Scientific	3	$10,000 +	Options: manual thresholding, auto thresholding (choose criterion based suited for data generally factorization), hysteresis thresholding (Thermo Scientific Avizo Software 9 User Guide).	Options: manual, threshold-based image segmentation with multi-thresholding or interactive thresholding, interactive top-hat (Thermo Scientific Avizo Software 9 User Guide).	Segmentation was completed with Avizo, but no quantified features were attributed to Avizo.
AutoQuant X3	Media Cybernetics	2	$7,000	Options: manual threshold, maximum/minimum thresholding (Media Cybernetics, Inc. User Manual Version X2)	OptiHistogram-BasedBased Segmentation (Media Cybernetics, Inc. User Manual Version X2)	Branch Ends (Huang et al., 2021), Branch Order (Huang et al., 2021), Count (Huang et al., 2021), Distance between Cells (Huang et al., 2021), Length (Huang et al., 2021)
Python	Python Software Foundation	2	Free	Coding language	Coding language	Coding language
Reconstruct	–	2	Custom software	–	–	–