The physics behind our software Jellyfish

When acquiring a single image using a confocal microscope, the result is the same if some of the light is attenuated by passing through the tissue, or if there is a decreased amount of fluorescent marker. It is impossible to tell these situations apart using a linear fluorescence measurements. However, the light intensity at the focus of confocal microscopes is high enough to cause fluorescence saturation. This means that the fluorescence output from the focus becomes non-linear in the intensity of excitation light. This non-linearity can be detected if we image with a special protocol with several different intensities of the excitation laser. By measuring the amount of fluorescence saturation, we can calculate how much light arrived at the focus. Using the Helmholtz-Stokes reversion reciprocity principle, and a small correction due to Stokes shift, we can from this also calculate how much emission light makes it back from the focus to the objective.

By knowing both how much excitation light is lost between the objective and the focus, and how much fluorescence light is being lost between the focus and the objective, we can correct confocal images for light loss. It is important that a laser that is strong enough to cause fluorescence saturation is used. In the case when the beam is diluted in many sub beams such as in spinning disc confocal imaging, it is beneficial to use a pulsed laser to achieve a sufficient amount of fluorescence saturation. This method is described in more detail in our patent WO2025029181A1.