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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.

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Spatial attenuation of fluorescein in cytoplasm and free solution. (a) xz section as shown through fluorescein in solution (100 µM), fluorescein conjugated at a latex sphere (10-µm-diam), and the latex sphere in the fluorescein solution. (b) Fluorescein-loaded HECV cell, (c) fluorescein-loaded PC3 cell, (d) GFP-expressing dictyostelium, and (e) fluorescein-loaded human neutrophil shown as phase-contrast image, xy and xz confocal planes. An enlarged version of the xz section of a polarized human neutrophil along the line marked is shown in e′. (f) A similar xz section is shown though two other neutrophils which have not yet “flattened out“ showing the clear attenuation of fluorescence toward the top of the cells. (g) The intensity profile of the free fluorescein and latex-attached fluorescein together with data from the cells shown, using the colored symbols indicated on the images. Bar: (a) 10 µm; (b–e) 25 µm
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fig3: Spatial attenuation of fluorescein in cytoplasm and free solution. (a) xz section as shown through fluorescein in solution (100 µM), fluorescein conjugated at a latex sphere (10-µm-diam), and the latex sphere in the fluorescein solution. (b) Fluorescein-loaded HECV cell, (c) fluorescein-loaded PC3 cell, (d) GFP-expressing dictyostelium, and (e) fluorescein-loaded human neutrophil shown as phase-contrast image, xy and xz confocal planes. An enlarged version of the xz section of a polarized human neutrophil along the line marked is shown in e′. (f) A similar xz section is shown though two other neutrophils which have not yet “flattened out“ showing the clear attenuation of fluorescence toward the top of the cells. (g) The intensity profile of the free fluorescein and latex-attached fluorescein together with data from the cells shown, using the colored symbols indicated on the images. Bar: (a) 10 µm; (b–e) 25 µm

Mentions: The organelle-free cytoplasmic zone of the pseudopod is optically “clear,” as opposed to the light-scattering and absorbing cytoplasm in the bulk of the cell (Fig. 2 c). This change in optical properties impacts the efficiency with which fluors are excited (Fig. 2 d). Mie scattering theory, which is an established basis for predicting the light-scattering effect of particles suspended within a medium (Mie 1908), predicts that light scattering by particles of the same order of magnitude as the wavelength of the incident light will be profound in some granular cells (Meyer, 1979: Ruban et al., 2007). In granular cytoplasm (granule diameters 0.2–0.3 µm), the attenuation coefficient could be as high as 700 mm−1 (Prahl, 2007), depending on the number of granules/volume of cytoplasm (see online supplemental material for details of a sample calculation, available at http://www.jcb.org/cgi/content/full/jcb.200806047/DC1). As a result of this attenuation, the efficiency of excitation of the fluor decreases as the imaging plane is taken from deeper within the cell. The effect of light scattering and absorbance can be seen in the orthogonal (z plane) image. In Fig. 3 a, z-plane images of fluorescein in free solution and immobilized on a latex sphere, an object with high light-scattering properties, are compared. The attenuation of signal by the latex sphere is obvious within a distance of only a few microns and a “shadow” of reduced excitation is cast into the free fluorescein above the sphere. For comparison, orthogonal plane images of soluble fluors within the cytoplasm of a motile neutrophil, Dictyostelium, epithelial cell (PC3) and endothelial cell (HECV) are shown (Fig. 3, b–e). Whereas the fluor in the latter cell types behave like fluor in free solution, in neutrophils attenuation is obvious (intermediate attenuation is observed in Dictyostelium). Therefore, it is expected that an optical artifact would be apparent in a pseudopodium imaged at optical planes of cells where z-plane attenuation is high.


Translocation or just location? Pseudopodia affect fluorescent signals.

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

Spatial attenuation of fluorescein in cytoplasm and free solution. (a) xz section as shown through fluorescein in solution (100 µM), fluorescein conjugated at a latex sphere (10-µm-diam), and the latex sphere in the fluorescein solution. (b) Fluorescein-loaded HECV cell, (c) fluorescein-loaded PC3 cell, (d) GFP-expressing dictyostelium, and (e) fluorescein-loaded human neutrophil shown as phase-contrast image, xy and xz confocal planes. An enlarged version of the xz section of a polarized human neutrophil along the line marked is shown in e′. (f) A similar xz section is shown though two other neutrophils which have not yet “flattened out“ showing the clear attenuation of fluorescence toward the top of the cells. (g) The intensity profile of the free fluorescein and latex-attached fluorescein together with data from the cells shown, using the colored symbols indicated on the images. Bar: (a) 10 µm; (b–e) 25 µm
© Copyright Policy - openaccess
Related In: Results  -  Collection

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

fig3: Spatial attenuation of fluorescein in cytoplasm and free solution. (a) xz section as shown through fluorescein in solution (100 µM), fluorescein conjugated at a latex sphere (10-µm-diam), and the latex sphere in the fluorescein solution. (b) Fluorescein-loaded HECV cell, (c) fluorescein-loaded PC3 cell, (d) GFP-expressing dictyostelium, and (e) fluorescein-loaded human neutrophil shown as phase-contrast image, xy and xz confocal planes. An enlarged version of the xz section of a polarized human neutrophil along the line marked is shown in e′. (f) A similar xz section is shown though two other neutrophils which have not yet “flattened out“ showing the clear attenuation of fluorescence toward the top of the cells. (g) The intensity profile of the free fluorescein and latex-attached fluorescein together with data from the cells shown, using the colored symbols indicated on the images. Bar: (a) 10 µm; (b–e) 25 µm
Mentions: The organelle-free cytoplasmic zone of the pseudopod is optically “clear,” as opposed to the light-scattering and absorbing cytoplasm in the bulk of the cell (Fig. 2 c). This change in optical properties impacts the efficiency with which fluors are excited (Fig. 2 d). Mie scattering theory, which is an established basis for predicting the light-scattering effect of particles suspended within a medium (Mie 1908), predicts that light scattering by particles of the same order of magnitude as the wavelength of the incident light will be profound in some granular cells (Meyer, 1979: Ruban et al., 2007). In granular cytoplasm (granule diameters 0.2–0.3 µm), the attenuation coefficient could be as high as 700 mm−1 (Prahl, 2007), depending on the number of granules/volume of cytoplasm (see online supplemental material for details of a sample calculation, available at http://www.jcb.org/cgi/content/full/jcb.200806047/DC1). As a result of this attenuation, the efficiency of excitation of the fluor decreases as the imaging plane is taken from deeper within the cell. The effect of light scattering and absorbance can be seen in the orthogonal (z plane) image. In Fig. 3 a, z-plane images of fluorescein in free solution and immobilized on a latex sphere, an object with high light-scattering properties, are compared. The attenuation of signal by the latex sphere is obvious within a distance of only a few microns and a “shadow” of reduced excitation is cast into the free fluorescein above the sphere. For comparison, orthogonal plane images of soluble fluors within the cytoplasm of a motile neutrophil, Dictyostelium, epithelial cell (PC3) and endothelial cell (HECV) are shown (Fig. 3, b–e). Whereas the fluor in the latter cell types behave like fluor in free solution, in neutrophils attenuation is obvious (intermediate attenuation is observed in Dictyostelium). Therefore, it is expected that an optical artifact would be apparent in a pseudopodium imaged at optical planes of cells where z-plane attenuation is high.

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