Fluorescence imaging artifacts accompanying pseudopodia formation
| Cellular condition | Consequence | Fluorescence Imaging Artifact |
| A. Non-uniformity of plasma membrane topography | ||
| (i) Membrane ruffling | ||
| (ii) Increased sub-resolution convolutions in pseudopodial membrane | Locally increased membrane within the imaging plane | Higher local fluorescence of membrane probes |
| (iii) Reduced sub-resolution convolutions in pseudopodial membrane | Locally reduced membrane within the imaging plane | Lower local fluorescence of membrane probes |
| B. Non-uniformity of pseudopodial z-plane thickness | Locally reduced cytoplasmic volume within the imaging plane | Lower fluorescence in “thinner” region of pseudopodium |
| (i) Extended pseudopodia thinner than non-pseudopodial regions | ||
| (ii) Thickening of termini relative of extending pseudopodia | Locally increased cytoplasmic volume within the imaging plane | Higher fluorescence localized to “thicker” regions of pseudopodium |
| (iii) z-plane extensions at pseudopodia termini not yet attached to substrate | ||
| C. Organelle exclusion from pseudopodiaa | ||
| (i) Reduced light scattering in extending pseudopodia (see Fig. 2 b) | (i) Locally increased excitation efficiency | |
| (ii) Reduced cytosolic density (see Fig. 2 a) | (ii) Locally increased cytoplasmic fluor concentration | Higher fluorescence in pseudopodia |
| Cellular condition | Consequence | Fluorescence Imaging Artifact |
| A. Non-uniformity of plasma membrane topography | ||
| (i) Membrane ruffling | ||
| (ii) Increased sub-resolution convolutions in pseudopodial membrane | Locally increased membrane within the imaging plane | Higher local fluorescence of membrane probes |
| (iii) Reduced sub-resolution convolutions in pseudopodial membrane | Locally reduced membrane within the imaging plane | Lower local fluorescence of membrane probes |
| B. Non-uniformity of pseudopodial z-plane thickness | Locally reduced cytoplasmic volume within the imaging plane | Lower fluorescence in “thinner” region of pseudopodium |
| (i) Extended pseudopodia thinner than non-pseudopodial regions | ||
| (ii) Thickening of termini relative of extending pseudopodia | Locally increased cytoplasmic volume within the imaging plane | Higher fluorescence localized to “thicker” regions of pseudopodium |
| (iii) z-plane extensions at pseudopodia termini not yet attached to substrate | ||
| C. Organelle exclusion from pseudopodiaa | ||
| (i) Reduced light scattering in extending pseudopodia (see Fig. 2 b) | (i) Locally increased excitation efficiency | |
| (ii) Reduced cytosolic density (see Fig. 2 a) | (ii) Locally increased cytoplasmic fluor concentration | Higher fluorescence in pseudopodia |
Artifacts listed in C have not previously been recognized and is the main subject of this paper.