Foci-Xpress: Automated and Fast Nuclear Foci Counting Tool

Abstract

In the nucleus, distinct, discrete spots or regions called "foci" have been identified, each harboring a specific molecular function. Accurate and efficient quantification of these foci is essential for understanding cellular dynamics and signaling pathways. In this study, we present an innovative automated image analysis method designed to precisely quantify subcellular foci within the cell nucleus. Manual foci counting methods can be tedious and time-consuming. To address these challenges, we developed an open-source software that automatically counts the number of foci from the indicated image files. We compared the foci counting efficiency, velocity, accuracy, and convenience of Foci-Xpress with those of other conventional methods in foci-induced models. We can adjust the brightness of foci to establish a threshold. The Foci-Xpress method was significantly faster than other conventional methods. Its accuracy was similar to that of conventional methods. The most significant strength of Foci-Xpress is automation, which eliminates the need for analyzing equipment while counting. This enhanced throughput facilitates comprehensive statistical analyses and supports robust conclusions from experiments. Furthermore, automation completely rules out biases caused by researchers, such as manual errors or daily variations. Thus, Foci-Xpress is a convincing, convenient, and easily accessible focus-counting tool for cell biologists.

Keywords: DNA damage; automated screening method; foci quantitative analysis; image processing; nuclear foci.

Figure 1

Overview of the software system: (A) workflow of Foci-Xpress; (B) main interface of Foci-Xpress.

Figure 2

Representative images of the working process of Foci-Xpress for capturing the nucleus in confocal microscopy images. (A) Image capture process for foci counting. The image represented merges DAPI and γH2AX. Files used by CZI estimated the regions of interest (ROI) value for the nuclei in the yellow border. Each nucleus is numbered to determine the number of nuclei. For foci counting, the channel of γH2AX images was converted into greyscale images. γH2AX foci in the nucleus were estimated based on the following parameters: nucleus size, foci intensity, and the number of foci in nuclei. Scale bar: 10 μm. Immunofluorescence was performed using a γH2AX foci (grren) and DAPI (blue). (B) Representative images of immunofluorescence (IF) performed on M3CTC-E1 cells. MC3T3-E1 cells were treated with 50 µM of H₂O₂ for 24 h. MC3T3-E1 cells stain γH2AX (phosphorylation of H2AX at ser139) in green (top image) and DAPI in blue (middle image). The image of the merged channels is shown at the bottom. Scale bar: 10 μm.

Figure 3

Analysis of damaged cells compared with classical methods. (A) Graph of the foci formation of γH2AX in cells treated with or without H₂O₂, as shown in Figure 2B. Error bars show the SEM of two duplicates, and ‘n.s’ indicates no significance. (B) Quantification of measurement time between the different counting methods. (C) The average number of γH2AX foci per cells for 100 cells treated with or without H₂O₂, as shown in Figure 2B. Error bars show the SEM from two duplicates and indicate a * p-value < 0.05.

Figure 4

Analysis of the time required for Foci-Xpress. A minimum of 10 images per group were randomly captured using a 40× objective lens following IF staining. The samples were imaged using a confocal microscope (LSM 800, Carl Zeiss). The same set of images was analyzed using three independent methods for each group: 5, 10, 20, 50, and 100 images.

Figure 5

Representative confocal microscopy images of TIF. (A) Representative colocalization images of TIF. MC3T3-E1 cells were treated with 50 µM of H₂O₂ for 24 h or were untreated. MC3T3-E1 cells were incubated with anti-γH2AX (phosphorylation of H2AX at ser139) in green (top image) and DAPI in blue (4th image from the top). Images of the middle and bottom are merged channels green + red or green + red + blue images. Scale bar: 10 μm. Immunofluorescence was performed using a γH2AX foci (grren), telemore probe (red) and DAPI (blue). (B) Representative colocalization of damaged cells with labeled γH2AX foci (green) and telomere DNA (red). Scale bar: 10 μm.

Figure 6

Graphs of colocalized foci and foci counting analysis at a single wavelength using Foci-Xpress. (A) Percentage of colocalization between γH2AX and the telomeric probe in the cells captured in Figure 5. The grey bars represent the H₂O₂ group, whereas the black bars represent the untreated control group. H₂O₂: cells treated with 50 µM of H₂O₂ for 24 h. (B) Percentage of γH2AX in the cells captured in Figure 5. The grey bars represent the H₂O₂ group, whereas the black bars represent the untreated control group. H₂O₂: cells treated with 50 µM of H₂O₂ for 24 h.