Figure 13.

Optical challenges during fluorescence imaging of ex ovo chick embryos

(A–C) Representative fields showing partial embryo views, uneven surface curvature, or embryonic structures within the field of view, leading to shading, depth-dependent signal attenuation, and focal plane variability.

(D–E) Fields containing both embryo and chorioallantoic membrane (CAM), illustrating heterogeneous optical properties and variable background fluorescence.

(F) Membrane curvature causes focal plane inconsistencies and reduced image sharpness across the field.

Ex ovo embryos exhibit heterogeneous optical properties and variable surface topography that can impair the detection of GFP-labeled tumor cells and Hoechst-stained NK cells.

To minimize artefacts, prioritize superficial regions with stable optical access, adjust illumination, apply flat-field correction or background subtraction, and exclude fields where uneven illumination or depth-related signal loss prevents reliable segmentation. Scale bar = 1 000 μm.