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Colocalization coefficients evaluating the distribution of molecular targets in microscopy methods based on pointed patterns

View Article: PubMed Central - PubMed

ABSTRACT

In biomedical studies, the colocalization is commonly understood as the overlap between distinctive labelings in images. This term is usually associated especially with quantitative evaluation of the immunostaining in fluorescence microscopy. On the other hand, the evaluation of the immunolabeling colocalization in the electron microscopy images is still under-investigated and biased by the subjective and non-quantitative interpretation of the image data. We introduce a novel computational technique for quantifying the level of colocalization in pointed patterns. Our approach follows the idea included in the widely used Manders’ colocalization coefficients in fluorescence microscopy and represents its counterpart for electron microscopy. In presented methodology, colocalization is understood as the product of the spatial interactions at the single-particle (single-molecule) level. Our approach extends the current significance testing in the immunoelectron microscopy images and establishes the descriptive colocalization coefficients. To demonstrate the performance of the proposed coefficients, we investigated the level of spatial interactions of phosphatidylinositol 4,5-bisphosphate with fibrillarin in nucleoli. We compared the electron microscopy colocalization coefficients with Manders’ colocalization coefficients for confocal microscopy and super-resolution structured illumination microscopy. The similar tendency of the values obtained using different colocalization approaches suggests the biological validity of the scientific conclusions. The presented methodology represents a good basis for further development of the quantitative analysis of immunoelectron microscopy data and can be used for studying molecular interactions at the ultrastructural level. Moreover, this methodology can be applied also to the other super-resolution microscopy techniques focused on characterization of discrete pointed structures.

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Localization of fibrillarin and PIP2 in the nucleoli in CM, SIM, and EM images with the selected image sections. a Fibrillarin and PIP2 detected by confocal fluorescence microscopy. b Localization of fibrillarin and PIP2 inspected by SIM. c Spatial distribution of PIP2 (black dots) and fibrillarin (red dots) in EM. FC—fibrillar centers, DFC—dense fibrillar component, N—nucleus, NL—nucleolus. Scale bars: 5 µm (A), 5 µm (B) and 100 nm (C)
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Fig3: Localization of fibrillarin and PIP2 in the nucleoli in CM, SIM, and EM images with the selected image sections. a Fibrillarin and PIP2 detected by confocal fluorescence microscopy. b Localization of fibrillarin and PIP2 inspected by SIM. c Spatial distribution of PIP2 (black dots) and fibrillarin (red dots) in EM. FC—fibrillar centers, DFC—dense fibrillar component, N—nucleus, NL—nucleolus. Scale bars: 5 µm (A), 5 µm (B) and 100 nm (C)

Mentions: The representative images obtained by CM, SIM, and EM are shown in Fig. 3. The EM image data were used to calculate the pair cross-correlation function (PCCF) shown as a graph in Fig. 4a. The levels of PCCF describe, how many times the measured frequency of particles exceeds the theoretical frequency, and they reveal the higher occurrence of the pairs of particles on the distance intervals less than 225 nm.Fig. 3


Colocalization coefficients evaluating the distribution of molecular targets in microscopy methods based on pointed patterns
Localization of fibrillarin and PIP2 in the nucleoli in CM, SIM, and EM images with the selected image sections. a Fibrillarin and PIP2 detected by confocal fluorescence microscopy. b Localization of fibrillarin and PIP2 inspected by SIM. c Spatial distribution of PIP2 (black dots) and fibrillarin (red dots) in EM. FC—fibrillar centers, DFC—dense fibrillar component, N—nucleus, NL—nucleolus. Scale bars: 5 µm (A), 5 µm (B) and 100 nm (C)
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC5037163&req=5

Fig3: Localization of fibrillarin and PIP2 in the nucleoli in CM, SIM, and EM images with the selected image sections. a Fibrillarin and PIP2 detected by confocal fluorescence microscopy. b Localization of fibrillarin and PIP2 inspected by SIM. c Spatial distribution of PIP2 (black dots) and fibrillarin (red dots) in EM. FC—fibrillar centers, DFC—dense fibrillar component, N—nucleus, NL—nucleolus. Scale bars: 5 µm (A), 5 µm (B) and 100 nm (C)
Mentions: The representative images obtained by CM, SIM, and EM are shown in Fig. 3. The EM image data were used to calculate the pair cross-correlation function (PCCF) shown as a graph in Fig. 4a. The levels of PCCF describe, how many times the measured frequency of particles exceeds the theoretical frequency, and they reveal the higher occurrence of the pairs of particles on the distance intervals less than 225 nm.Fig. 3

View Article: PubMed Central - PubMed

ABSTRACT

In biomedical studies, the colocalization is commonly understood as the overlap between distinctive labelings in images. This term is usually associated especially with quantitative evaluation of the immunostaining in fluorescence microscopy. On the other hand, the evaluation of the immunolabeling colocalization in the electron microscopy images is still under-investigated and biased by the subjective and non-quantitative interpretation of the image data. We introduce a novel computational technique for quantifying the level of colocalization in pointed patterns. Our approach follows the idea included in the widely used Manders’ colocalization coefficients in fluorescence microscopy and represents its counterpart for electron microscopy. In presented methodology, colocalization is understood as the product of the spatial interactions at the single-particle (single-molecule) level. Our approach extends the current significance testing in the immunoelectron microscopy images and establishes the descriptive colocalization coefficients. To demonstrate the performance of the proposed coefficients, we investigated the level of spatial interactions of phosphatidylinositol 4,5-bisphosphate with fibrillarin in nucleoli. We compared the electron microscopy colocalization coefficients with Manders’ colocalization coefficients for confocal microscopy and super-resolution structured illumination microscopy. The similar tendency of the values obtained using different colocalization approaches suggests the biological validity of the scientific conclusions. The presented methodology represents a good basis for further development of the quantitative analysis of immunoelectron microscopy data and can be used for studying molecular interactions at the ultrastructural level. Moreover, this methodology can be applied also to the other super-resolution microscopy techniques focused on characterization of discrete pointed structures.

No MeSH data available.