FISH signal counting and ratios
FISH shows where fluorescent probes bind as spots. Counting target-gene signals and control signals from the same chromosome in each nucleus tells you whether a gene is gained (amplified) or lost (deleted).
Upload an image — the signal colors present are detected and suggested, then counts, ratios and HSRs are computed.
Open the FISH tool →1. Why a control probe and a ratio
A cell with extra copies of the whole chromosome (ploidy change) also shows more target signals. Dividing by a control probe on the same chromosome (e.g. a centromere probe) separates chromosome gain from true gene amplification.
2. Example
| Nuclei counted | Target total | Control total | Ratio | Mean target per nucleus |
|---|---|---|---|---|
| 20 | 88 | 40 | 2.2 | 4.4 |
Report the mean signals per nucleus alongside the ratio. Cut-offs differ by assay and probe, so clinical interpretation must follow that assay’s guidelines; this guide and tool support research quantification.
3. Counting rules
- Count only non-overlapping nuclei with intact borders; skip cut or overlapping nuclei.
- Fix the number of nuclei in advance (typically 20–100) and spread them across several fields.
- Only spots of similar size and shape count; two spots closer than about one signal diameter are usually counted as one.
- Tight clusters (HSRs) cannot be counted individually — record them separately as estimates.
4. Multi-color probes and overlaps
Red and green signals that overlap look yellow (fusion). With translocation probes (fusion or break-apart), that overlap or separation is exactly what you read. With extra colors such as aqua or gold, make sure blended edges are not counted as another color.
A red signal on a blue DAPI nucleus can look pink on screen. Counting on per-channel originals rather than a merged image reduces this confusion (exporting microscope images).
Upload an image — the signal colors present are detected and suggested, then counts, ratios and HSRs are computed.
Open the FISH tool →