Limits...
Translocation or just location? Pseudopodia affect fluorescent signals.

Dewitt S, Darley RL, Hallett MB - J. Cell Biol. (2009)

Bottom Line: Localized increases in the signal from cytosolic fluorescent protein constructs, for example, are frequently used as evidence for translocation of proteins to specific sites within the cell.However, differences in optical and geometrical properties of cytoplasm can influence the recorded intensity of the probe signal.Pseudopodia are especially problematic because their cytoplasmic properties can cause abrupt increases in fluorescent signal of both GFP and fluorescein.

View Article: PubMed Central - PubMed

Affiliation: Neutrophil Signalling Group, School of Medicine, Cardiff University, Heath Park, Cardiff, Wales, UK.

ABSTRACT
The use of fluorescent probes is one of the most powerful techniques for gaining spatial and temporal knowledge of dynamic events within living cells. Localized increases in the signal from cytosolic fluorescent protein constructs, for example, are frequently used as evidence for translocation of proteins to specific sites within the cell. However, differences in optical and geometrical properties of cytoplasm can influence the recorded intensity of the probe signal. Pseudopodia are especially problematic because their cytoplasmic properties can cause abrupt increases in fluorescent signal of both GFP and fluorescein. Investigators should therefore be cautious when interpreting fluorescence changes within a cell, as these can result from either translocation of the probe or changes in the optical properties of the milieu surrounding the probe.

Show MeSH

Related in: MedlinePlus

Principle of spatial-optical effect at leading pseudopodium. (a) Phase-contrast image of polarized human neutrophils showing the leading pseudopodium of the cell marked “ps” and the uropod at the rear marked “U”. The granular and organelle-free zones are marked for clarity. (b) A “sideways” or orthogonal view of the cell showing how the amount of organelle-free cytoplasm, and hence the fluor gross concentration and optical path-length (PL) available, varies along the length of the cell. (c) The differential effect of light scattering by granular zone of cytoplasm and transmission in the clear zone are illustrated in this orthogonal view. (d) The predicted difference in efficiency of fluorescence excitation along the axis of the cell. Bar (a), 5 µm
© Copyright Policy - openaccess
Related In: Results  -  Collection

License 1 - License 2
getmorefigures.php?uid=PMC2654297&req=5

fig2: Principle of spatial-optical effect at leading pseudopodium. (a) Phase-contrast image of polarized human neutrophils showing the leading pseudopodium of the cell marked “ps” and the uropod at the rear marked “U”. The granular and organelle-free zones are marked for clarity. (b) A “sideways” or orthogonal view of the cell showing how the amount of organelle-free cytoplasm, and hence the fluor gross concentration and optical path-length (PL) available, varies along the length of the cell. (c) The differential effect of light scattering by granular zone of cytoplasm and transmission in the clear zone are illustrated in this orthogonal view. (d) The predicted difference in efficiency of fluorescence excitation along the axis of the cell. Bar (a), 5 µm

Mentions: Fluorescence imaging artifacts accompanying pseudopodia formation


Translocation or just location? Pseudopodia affect fluorescent signals.

Dewitt S, Darley RL, Hallett MB - J. Cell Biol. (2009)

Principle of spatial-optical effect at leading pseudopodium. (a) Phase-contrast image of polarized human neutrophils showing the leading pseudopodium of the cell marked “ps” and the uropod at the rear marked “U”. The granular and organelle-free zones are marked for clarity. (b) A “sideways” or orthogonal view of the cell showing how the amount of organelle-free cytoplasm, and hence the fluor gross concentration and optical path-length (PL) available, varies along the length of the cell. (c) The differential effect of light scattering by granular zone of cytoplasm and transmission in the clear zone are illustrated in this orthogonal view. (d) The predicted difference in efficiency of fluorescence excitation along the axis of the cell. Bar (a), 5 µm
© Copyright Policy - openaccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC2654297&req=5

fig2: Principle of spatial-optical effect at leading pseudopodium. (a) Phase-contrast image of polarized human neutrophils showing the leading pseudopodium of the cell marked “ps” and the uropod at the rear marked “U”. The granular and organelle-free zones are marked for clarity. (b) A “sideways” or orthogonal view of the cell showing how the amount of organelle-free cytoplasm, and hence the fluor gross concentration and optical path-length (PL) available, varies along the length of the cell. (c) The differential effect of light scattering by granular zone of cytoplasm and transmission in the clear zone are illustrated in this orthogonal view. (d) The predicted difference in efficiency of fluorescence excitation along the axis of the cell. Bar (a), 5 µm
Mentions: Fluorescence imaging artifacts accompanying pseudopodia formation

Bottom Line: Localized increases in the signal from cytosolic fluorescent protein constructs, for example, are frequently used as evidence for translocation of proteins to specific sites within the cell.However, differences in optical and geometrical properties of cytoplasm can influence the recorded intensity of the probe signal.Pseudopodia are especially problematic because their cytoplasmic properties can cause abrupt increases in fluorescent signal of both GFP and fluorescein.

View Article: PubMed Central - PubMed

Affiliation: Neutrophil Signalling Group, School of Medicine, Cardiff University, Heath Park, Cardiff, Wales, UK.

ABSTRACT
The use of fluorescent probes is one of the most powerful techniques for gaining spatial and temporal knowledge of dynamic events within living cells. Localized increases in the signal from cytosolic fluorescent protein constructs, for example, are frequently used as evidence for translocation of proteins to specific sites within the cell. However, differences in optical and geometrical properties of cytoplasm can influence the recorded intensity of the probe signal. Pseudopodia are especially problematic because their cytoplasmic properties can cause abrupt increases in fluorescent signal of both GFP and fluorescein. Investigators should therefore be cautious when interpreting fluorescence changes within a cell, as these can result from either translocation of the probe or changes in the optical properties of the milieu surrounding the probe.

Show MeSH
Related in: MedlinePlus