FISHji: new ImageJ macros for the quantification of fluorescence in epifluorescence images

Fluorescence in situ hybridization (FISH) is based on the use of fluorescent staining dyes, however, the signal intensity of the images obtained by microscopy is seldom quantified with accuracy by the researcher. The development of innovative digital image processing programs and tools has been trying to overcome this problem, however, the determination of fluorescent intensity in microscopy images still has issues due to the lack of precision in the results and the complexity of existing software. This work presents FISHji, a set of new ImageJ methods for automated quantification of fluorescence in images obtained by epifluorescence microscopy. To validate the methods, results obtained by FISHji were compared with results obtained by flow cytometry. The mean correlation between FISHji and flow cytometry was high and significant, showing that the imaging methods are able to accurately assess the signal intensity of fluorescence images. FISHji are available for non-commercial use at http://paginas.fe.up.pt/nazevedo/.

This work was financially supported by: Project UID/EQU/00511/2013-LEPABE, by the FCT/MEC with national funds and when applicable co-funded by FEDER in the scope of the P2020 Partnership Agreement; Project NORTE-07-0124-FEDER-000025—RL2_Environment&Health, by FEDER funds through Programa Operacional Factores de Competitividade—COMPETE, by the Programa Operacional do Norte (ON2) program and by national funds through FCT—Fundação para a Ciência e a Tecnologia; DNA mimics Research Project PIC/IC/82815/2007, PhD Fellowship SFRH/BD/72999/2010 and Post-Doctoral fellowship SFRH/BPD/78846/2011.