imaris intensity based colocalization analysis Search Results


colocalization analysis  (Oxford Instruments)
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Oxford Instruments colocalization analysis
MIM/IMD-L localizes to the plasma membrane in filopodia and associates actin to membranes in vitro. (A) GFP–MIM/IMD-L localized to the plasma membrane and coated the F-actin bundles in filopodia of U2OS cell. 3D analysis derived from seven confocal planes shows F-actin in red (all panels), MIM/IMD in green (right), and <t>colocalization</t> in yellow (middle and right). Colocalization area (built by ImarisColoc software) is only a thin layer between actin and MIM/IMD and is almost invisible when the three channels are merged (right), as the MIM/IMD signal at the plasma membrane covers the interior of filopodia. Bar, 1 μm. (B) In vitro light microscopy assay. 1 μM F-actin (50% Alexa 568–labeled), 2.5 μM MIM/IMD-L, and 1.67 μM lipid vesicles (0/30% PI[4,5]P 2 ) were mixed and applied on polyornithine-coated glass slides for imaging. In the presence of MIM/IMD-L and PI(4,5)P 2 , actin concentrated at the sites of vesicles, indicating MIM/IMD-L–associating actin and vesicles (top). MIM/IMD-L with vesicles without PI(4,5)P 2 or MIM/IMD-L Mut15, which displays defects in actin and PI(4,5)P 2 binding, induced much weaker colocalization between actin and vesicles. Red arrowheads indicate vesicles that show clear colocalization with actin, and green arrowheads indicate vesicles without colocalizing actin. Bar, 20 μm. (C) The intensity of the actin staining on the vesicles was quantified from 19–20 randomly selected vesicles. Error bars indicate SEM values. A t test was used to analyze p-values and showed statistically significant differences (***, P < 0.001; **, P < 0.01).
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MIM/IMD-L localizes to the plasma membrane in filopodia and associates actin to membranes in vitro. (A) GFP–MIM/IMD-L localized to the plasma membrane and coated the F-actin bundles in filopodia of U2OS cell. 3D analysis derived from seven confocal planes shows F-actin in red (all panels), MIM/IMD in green (right), and colocalization in yellow (middle and right). Colocalization area (built by ImarisColoc software) is only a thin layer between actin and MIM/IMD and is almost invisible when the three channels are merged (right), as the MIM/IMD signal at the plasma membrane covers the interior of filopodia. Bar, 1 μm. (B) In vitro light microscopy assay. 1 μM F-actin (50% Alexa 568–labeled), 2.5 μM MIM/IMD-L, and 1.67 μM lipid vesicles (0/30% PI[4,5]P 2 ) were mixed and applied on polyornithine-coated glass slides for imaging. In the presence of MIM/IMD-L and PI(4,5)P 2 , actin concentrated at the sites of vesicles, indicating MIM/IMD-L–associating actin and vesicles (top). MIM/IMD-L with vesicles without PI(4,5)P 2 or MIM/IMD-L Mut15, which displays defects in actin and PI(4,5)P 2 binding, induced much weaker colocalization between actin and vesicles. Red arrowheads indicate vesicles that show clear colocalization with actin, and green arrowheads indicate vesicles without colocalizing actin. Bar, 20 μm. (C) The intensity of the actin staining on the vesicles was quantified from 19–20 randomly selected vesicles. Error bars indicate SEM values. A t test was used to analyze p-values and showed statistically significant differences (***, P < 0.001; **, P < 0.01).

Journal: The Journal of Cell Biology

Article Title: Missing-in-metastasis and IRSp53 deform PI(4,5)P 2 -rich membranes by an inverse BAR domain–like mechanism

doi: 10.1083/jcb.200609176

Figure Lengend Snippet: MIM/IMD-L localizes to the plasma membrane in filopodia and associates actin to membranes in vitro. (A) GFP–MIM/IMD-L localized to the plasma membrane and coated the F-actin bundles in filopodia of U2OS cell. 3D analysis derived from seven confocal planes shows F-actin in red (all panels), MIM/IMD in green (right), and colocalization in yellow (middle and right). Colocalization area (built by ImarisColoc software) is only a thin layer between actin and MIM/IMD and is almost invisible when the three channels are merged (right), as the MIM/IMD signal at the plasma membrane covers the interior of filopodia. Bar, 1 μm. (B) In vitro light microscopy assay. 1 μM F-actin (50% Alexa 568–labeled), 2.5 μM MIM/IMD-L, and 1.67 μM lipid vesicles (0/30% PI[4,5]P 2 ) were mixed and applied on polyornithine-coated glass slides for imaging. In the presence of MIM/IMD-L and PI(4,5)P 2 , actin concentrated at the sites of vesicles, indicating MIM/IMD-L–associating actin and vesicles (top). MIM/IMD-L with vesicles without PI(4,5)P 2 or MIM/IMD-L Mut15, which displays defects in actin and PI(4,5)P 2 binding, induced much weaker colocalization between actin and vesicles. Red arrowheads indicate vesicles that show clear colocalization with actin, and green arrowheads indicate vesicles without colocalizing actin. Bar, 20 μm. (C) The intensity of the actin staining on the vesicles was quantified from 19–20 randomly selected vesicles. Error bars indicate SEM values. A t test was used to analyze p-values and showed statistically significant differences (***, P < 0.001; **, P < 0.01).

Article Snippet: The confocal image stacks were deconvoluted with AutoQuant AutoDeblur 3D Blind Deconvolution (AutoQuant Imaging, Inc.), and the 3D reconstructions and the colocalization analysis were made with Bitplane Imaris (Bitplane Inc.).

Techniques: Membrane, In Vitro, Derivative Assay, Software, Light Microscopy, Labeling, Imaging, Binding Assay, Staining