Foci counting gets treated as a single-channel problem. One marker, one condition, a count per cell. That answers part of the question.

The biology almost always involves more than one marker. DNA damage brings γH2AX and 53BP1 together at the same site. Replication stress changes the ratio of licensed to active origins. Co-localisation of two foci marks a specific molecular event that neither marker captures alone.

The problem compounds quickly. Count foci per cell in channel one, count them in channel two, then determine which foci from each channel overlap, per cell, across thousands of cells, across multiple conditions. Most pipelines handle one channel and approximate the rest.

In Cytely, each channel is segmented independently and foci are detected as individual objects. Association is then computed per cell across as many markers as the experiment requires: individual counts per channel, plus every combination of co-localising foci. Comparing those values across conditions becomes a standard gating exercise.

How the assay runs

Segment cells → detect foci in Ch2 and Ch3 independently → count each type per cell and compute ratios. To determine which individual foci from marker A are positive for marker B: detect spots in Ch2 and measure Ch3 intensity in those spots.

What you provide

  • Ch1: DAPI
  • Ch2: foci marker A
  • Ch3: foci marker B

What you get

  • Foci count per channel per cell
  • Count ratio of marker A to marker B per cell
  • % cells positive for both markers

Ready to run

Telomere-associated DNA damage foci (γH2AX + telomere FISH), autophagosome-mitochondria association (LC3 + TOM20 for mitophagy), dual-probe FISH co-expression (% cells expressing gene A and gene B), PLA + cell type marker, any two-channel puncta comparison.

Inquire for support

Per-spot spatial distance measurement between two spot sets, 3D spot association.

Explore the Multi-Marker Foci Association assay →