## 4 Conclusions  
This work provides an extensive overview of open-source tools today available for researchers interested in estimating the volume of multicellular 3D-_oids_, besides presenting a new version of _ReViMS_ ([[http://sourceforge.net/p/revims](http://sourceforge.net/p/revims)]([http://sourceforge.net/p/revims](http://sourceforge.net/p/revims))), a user-friendly software tool specifically designed to estimate the volume of organoids and, in general, multicellular _aggregates_ with irregular shape.  
Using multicellular cancer _aggregates_ imaged by a light-sheet fluorescent microscope, we compared different open-source tools for measuring the culture volume by starting from microscope-acquired images.  
First of all, we proved that even an expert operator provides slightly different segmentations if asked to segment two times the same blurry fluorescence images. Accordingly, in case of fluorescent datasets, the Ground Truth of the object's border is characterised by limited robustness and algorithms for combining different segmentations whilst removing outliers are needed. Then, depending on the geometry of the 3D in vitro model considered (i.e., spherical or irregular shape), solutions based on a single projection (e.g., _ReViSP_) or suites analysing different sections (e.g., _ReViMS_ and _MITK_) can be used to estimate the volume with different accuracy. In particular, we proved that considering a single projection works just in case of _spheroids_ (i.e., multicellular _aggregates_ with a spherical shape) and very inaccurate measurements are obtained in case of general cell _aggregates_ with irregular shape. Semi-automatic approaches like _MITK_ generally provide good results also in case of cell _aggregates_, but the accuracy of the measures strongly depend on the number of initial manually segmented images. However, segmentation errors affect the accuracy of the final measurement. Eventually, software like _OpenSegSPIM_, which automatically outlines the single cells of multicellular _aggregates_, can be used to obtain the volume of the entire culture by summing the single terms. However, the fairness of estimation logically depends on the extent by which cells overlap. _ReViMS_ represents today the best trade-off between segmentation accuracy and computational time required in case of volume estimation of multicellular _aggregates_ characterised by an irregular shape.  
All the images and segmentations considered in this work, useful for instance for testing other volume estimation tools, are freely provided at: www.filippopiccinini.it/FP/2019 FP-LSFM.zip.  
Conceptualization, I.D.S., E.T. and F.P.; Data curation, F.P.; Formal analysis, E.T. and F.P.; Investigation, I.D.S., E.T. and F.P.; Project administration, F.P.; Software, F.P.; Supervision, P.H. and A.B.; Validation, F.P.; Writing--original draft, I.D.S. and F.P.; Writing--review and editing, E.T. All authors read and approved the final manuscript.  
F.P., E.T., and P.H. acknowledge HAS-LENDULET-BIOMAG and the European Union and the European Regional Development Funds (GINOP-2.3.2-15-2016-00006, GINOP-2.3.2-15-2016-00026). F.P. acknowledges support from the NEUBIAS, COST Action (European Cooperation in Science and Technology) CA15124, for a granted short term scientific mission (ref: 41037) paving the way for this work.  
We would like to thank [PERSON] (Institute of Molecular and Cell Biology, IMCB--A*STAR, Singapore) and the other people of _OpenSegSPIM_ for suggestions and help to use their tool; [PERSON] (University of Antwerp, Belgium) for helping implement the 3D extension of the STAPLE algorithm; and [PERSON] and [PERSON] (Biological Research Centre, Szeged, Hungary) for sharing with us important opinions about the tools.  
The authors declare no conflict of interest.