Materials

1. Citations

Small Fish is an open source software pusblished under BSD 2-Clause License and requires credit to the original project. Small Fish is in a publication process but for now you can cite this project using the permenant archive (Zeonodo.org) DOI : https://doi.org/10.5281/zenodo.15124657

Floric Slimani. (2026). SmallFishGUI/small_fish_gui A User-Friendly Graphical Interface for smFISH Image Quantification (Version v2.3.2) [Computer software]. doi: https://doi.org/10.5281/zenodo.15124657

This project is based on published work that also require citations.

Cellpose2 cit:
version < 2.0.1
Stringer, C., Wang, T., Michaelos, M., & Pachitariu, M. (2021). Cellpose: a generalist algorithm for cellular segmentation. Nature methods, 18(1), 100-106.
version >= 2.0.1
Pachitariu, M., Rariden, M., & Stringer, C. (2025). Cellpose-SAM: superhuman generalization for cellular segmentation. bioRxiv.

BigFish cit:
Imbert A, Ouyang W, Safieddine A, Coleno E, Zimmer C, Bertrand E, Walter T, Mueller F. FISH-quant v2: a scalable and modular tool for smFISH image analysis. RNA. 2022 Jun;28(6):786-795. doi: 10.1261/rna.079073.121. Epub 2022 Mar 28. PMID: 35347070; PMCID: PMC9074904.

2. Material&Methods example

Example

You can adapt the following example to your usage of the software and your parameters.

Image quantifications were based SmallFish an open-source software including an image analysis pipeline that included cell segmentation, threshold-based spot detection, dense region deconvolution, and a Density-Based Spatial Clustering of Applications with Noise (DBSCAN) algorithm. Firstly, nuclei and cytoplasm segmentation were inferred in 2D on image stack mean projection using Cellpose neural network74. Secondly, single molecules were detected as 3D spots using the Big-FISH package75, which relies on an intensity Laplacian of Gaussian (LoG) filter followed by a local maxima filter and a user-adjusted threshold. During analysis, we ensured that the same thresholds were applied to images made for the same RNA, for all cell treatments. As local maxima detection can lead to a misestimation of single molecule number in bright regions where individual molecules cannot be distinguished, a dense region deconvolution step was added. It consists of fitting the median detected spot with a 3D-gaussian to construct a reference spot and then reconstructing the bright region signal using multiple reference spots, thus providing the number of single RNAs in bright regions. Thirdly, a DBSCAN was used to assign spots to clusters (foci). At this point, detected spots were divided in clustered spots (spots assigned to a cluster) or free spots. Finally, a centroid calculation was performed to assign coordinates to each cluster. This single-molecule quantification process was repeated on all smFISH or SunTag channels, enabling quantification of RNA colocalization. During analysis, filtering was used to discard aberrations: (i) cells without nucleus or with more than one nuclei; (ii) any cell whose mask laid on the border of the field of view and that was thus considered incomplete; (iii) any spot detected outside of segmented cells. Distances between spots were computed using the exact Euclidian distance. Finally, spots were considered colocalizing when found closer together than a cutoff distance of 310 nm.

Additional informations on bias during analysis :

• During cell segmentation both cytoplasm and nucleus are segmented, though when a cytoplasm was found without nucleus or with more than one nuclei the cell was discarded.

• Any cell whose mask lies on the border of the field of view was considered incomplete and thus was discarded.

• Any spot detected outside of cell segmentations was discarded for cell quantification.

3. Putting code at the disposal of journal during publication

Journals should ask you to put with your publication the code you used to quantify your datasets in an immutable way so you can not alter post publication the quantification pipeline and other people can still reproduce your results. However Small Fish gui is an open source project that might keep evolving on github, so, to adress this issue, starting from 1.9.3 version (March 2025) permanent releases will be published on zenodo as it is broadly accepted by scientific journals for code publications and supports versioning.

To properly share your quantification, add a reference to Small Fish DOI link : https://doi.org/10.5281/zenodo.15124658, the parameters you used for quantifications (always available in the result) file and tag along the version of the software you are using : available in init.py file.