## 3 Results and Discussion
### Defining the Volume-Estimation Tool  
Figure 4 reports a flow-chart intended to help biologists, and researchers in general, to define an appropriate open-source tool to estimate the volume of multicellular _aggregates_ according to the characteristics of the used 3D in vitro model; Table 3 summarises the main features of the suggested tools, according to the template table proposed in [28]. If the cell _aggregate_ has a nearly spherical shape (i.e., it is a _spheroid_) and there is no interest in considering the single volumes of the cells composing the _aggregate_, _ReViSP_ is the suggested open-source tool, as it just requires one single projection of the _spheroid_ (i.e., a 2D image), easily attainable with a standard brightfield, phase-contrast, or DIC microscope without need of fluorescence staining. When, in the interest of estimating the total volume of the cell _aggregate_ also considering statistics about the volumes of the composing single cells, a 3D image is required, which is easily attainable with a confocal/multiphoton/LSFM. In this case, the tool \begin{table}
\begin{tabular}{l c c c c c} \hline \hline  & **Ground Truth Volume** & _ReViMS_ & _MITK_ & _OpenSegSPIM_ & _ReViMS_ & _MITK_ & _OpenSegSPIM_ \\  & **Volume** & **Volume** & **Volume** & **Volume** & **Volume** (\(\mu\)m\({}^{3}\))** & **AD%** & **AD%** & **AD%** \\  & **(\(\mu\)m\({}^{3}\))** & **(\(\mu\)m\({}^{3}\))** & **(\(\mu\)m\({}^{3}\))** & **(\(\mu\)m\({}^{3}\))** & **(\(\mu\)m\({}^{3}\))** & **(\(\mu\)m\({}^{3}\))** & **(\(\mu\)m\({}^{3}\))** \\ \hline MA1 & 13.418.918,238 & 13.294.691,377 & 13.742.255,460 & 11.484.703,264 & 0,9 & 2,4 & 14,4 \\ MA2 & 15.101.283,699 & 15.265.923,983 & 15.015.987,327 & 14.034.452,449 & 1,1 & 0,6 & 7,1 \\ MA3 & 11.619.907,849 & 11.658.164,658 & 11.569.970,771 & 11.000.449,993 & 0,3 & 0,4 & 5,3 \\ \hline \hline \end{tabular}
\end{table} 
Table 2: Accuracy of _MITK_ and _OpenSegSPIM_ volume estimations. we suggest is _OpenSegSPIM_ or _MITK_. In case the used 3D in vitro model is not a _spheroid_ (i.e., it is characterised by an irregular shape) and the researcher is only interested in the total volume of the _aggregate_, _ReViMS_ is the tool we suggest using. \begin{table}
\begin{tabular}{c c c c c} \hline \hline  & _ReViSP_ **(Version 2.1)** & _ReViMS_ **(Version 2.0)** & \
\begin{tabular}{c} _MITK_ **(Version** \\ **2018.04)** \\ \end{tabular} &
\begin{tabular}{c} _OpenSegSPIM_ **(Version** \\ **1.1)** \\ \end{tabular} \\ \hline \hline DOCUMENTATION & & & & \\ \hline User guide & & & & \\ Website & & & & \\ Video tutorial & & & & \\ Open source code & & & & \\ Implementation language & MATLAB & MATLAB & C++ & MATLAB \\ Test dataset/demo & & & & \\ Link to code/executable & sourceforge.net/p/revisp & sourceforge.net/p/revimus & mikik.org & opensegpim.weebly.com \\ Scientific reference & [PERSON] et al., 2015 [PERSON] et al., 2017 [PERSON] et al., 2017 12 [PERSON] et al., 2005 [PERSON] et al., 2005 22 & [PERSON] et al., 2016 11 \\ \hline USABILITY & & & & \\ \hline No programming & & & & \\ experience required & & & & \\ User-friendly GUI & & & & \\ Intuitive visualization & & & & \\ settings & & & & \\ Does not require & & & & \\ commercial licence & & & & \\ Portability on & & & & \\ Win/Linux/Mac & Win & Win/Linux & & Win/Mac \\ \hline FUNCTIONNALITY & & & & \\ \hline Designed for multicellular & & & & \\ aggregates & & & & \\ Automatic aggregate & & & & \\ segmentation & & & & \\ Automatic single-cell & & & & \\ segmentation & & & & \\ Does not require human & & & & \\ interaction & & & & \\ \hline OUTPUT & & & & \\ \hline
3D rendering & & & & \\ Volume statistics & & & & \\ other features statistics & & & & \\ \hline \hline \end{tabular} \(\bullet\), available/yes; o, not available/no  
\end{table} 
Table 3: Suggested volume-estimation tools comparison. Figure 4: Flow-chart helping to define an appropriate open-source tool according to the characteristics of the used 3D in vitro model. _ReViSP_ can be used just to estimate the total volume of a _spheroid_ and it requires a single 2D image as input.