Limits...
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.

No MeSH data available.


Conceptual comparison of EM colocalization coefficients versus the overlap in FM. a The colocalization in EM is based on the single-particle colocalization resulting in colocalizing pairs of particles on the given distance range. On the contrary, FM colocalization is based on the overlapping signals in pixels from the different color channels. b The relative frequency distribution of the colocalizing/non-colocalizing particles/pixels of the model examples in (A). After combining the information about the colocalizing particles, we conclude the additive characteristics of EM colocalization versus nonadditive but union characteristics of FM colocalization. Additionally, the first two bars for labels A and B in the bar graph from EM image include the relations between proposed coefficients:  and  (Eqs. 11, 12). c The Manders’ overlap coefficients in FM are calculated based on the intersection of the signals (overlap) and the union of the overlapped plus non-overlapped pixels of the labels in (A). The Manders’ overlap coefficient M1 for the label A can be alternatively calculated as division of the proportions (4/32)/(24/32) = 4/24, that is, 12.5 %/75.0 % ≈ 16.7 %. Coefficient M2 for the label B is: (4/32)/(12/32) = 4/12, that is, 12.5 %/37.5 % ≈ 33.3 %. The EM relative aggregated colocalization coefficients  for the label A and  for the label B are calculated in the similar manner from the frequency distribution of the colocalizing and non-colocalizing particles of the corresponding type (Eq. 4). Also, the alternative calculation is based on the identical approach as in FM. On the other hand, we can characterize more precisely this ratio using our proposed definitions:  and  (Eqs. 13, 14) based on the data from (A) and (B)
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC5037163&req=5

Fig1: Conceptual comparison of EM colocalization coefficients versus the overlap in FM. a The colocalization in EM is based on the single-particle colocalization resulting in colocalizing pairs of particles on the given distance range. On the contrary, FM colocalization is based on the overlapping signals in pixels from the different color channels. b The relative frequency distribution of the colocalizing/non-colocalizing particles/pixels of the model examples in (A). After combining the information about the colocalizing particles, we conclude the additive characteristics of EM colocalization versus nonadditive but union characteristics of FM colocalization. Additionally, the first two bars for labels A and B in the bar graph from EM image include the relations between proposed coefficients: and (Eqs. 11, 12). c The Manders’ overlap coefficients in FM are calculated based on the intersection of the signals (overlap) and the union of the overlapped plus non-overlapped pixels of the labels in (A). The Manders’ overlap coefficient M1 for the label A can be alternatively calculated as division of the proportions (4/32)/(24/32) = 4/24, that is, 12.5 %/75.0 % ≈ 16.7 %. Coefficient M2 for the label B is: (4/32)/(12/32) = 4/12, that is, 12.5 %/37.5 % ≈ 33.3 %. The EM relative aggregated colocalization coefficients for the label A and for the label B are calculated in the similar manner from the frequency distribution of the colocalizing and non-colocalizing particles of the corresponding type (Eq. 4). Also, the alternative calculation is based on the identical approach as in FM. On the other hand, we can characterize more precisely this ratio using our proposed definitions: and (Eqs. 13, 14) based on the data from (A) and (B)

Mentions: To describe the colocalization situation more detailed, we derive another important statistical coefficients, which includes the integration of the first and second level coefficients. By this combination, we receive the set of four summary measures, which includes the complex information about the proportions of colocalizing particles (, ) and non-colocalizing particles (, ) of the given labeling on the average image (Fig. 1):Fig. 1


Colocalization coefficients evaluating the distribution of molecular targets in microscopy methods based on pointed patterns
Conceptual comparison of EM colocalization coefficients versus the overlap in FM. a The colocalization in EM is based on the single-particle colocalization resulting in colocalizing pairs of particles on the given distance range. On the contrary, FM colocalization is based on the overlapping signals in pixels from the different color channels. b The relative frequency distribution of the colocalizing/non-colocalizing particles/pixels of the model examples in (A). After combining the information about the colocalizing particles, we conclude the additive characteristics of EM colocalization versus nonadditive but union characteristics of FM colocalization. Additionally, the first two bars for labels A and B in the bar graph from EM image include the relations between proposed coefficients:  and  (Eqs. 11, 12). c The Manders’ overlap coefficients in FM are calculated based on the intersection of the signals (overlap) and the union of the overlapped plus non-overlapped pixels of the labels in (A). The Manders’ overlap coefficient M1 for the label A can be alternatively calculated as division of the proportions (4/32)/(24/32) = 4/24, that is, 12.5 %/75.0 % ≈ 16.7 %. Coefficient M2 for the label B is: (4/32)/(12/32) = 4/12, that is, 12.5 %/37.5 % ≈ 33.3 %. The EM relative aggregated colocalization coefficients  for the label A and  for the label B are calculated in the similar manner from the frequency distribution of the colocalizing and non-colocalizing particles of the corresponding type (Eq. 4). Also, the alternative calculation is based on the identical approach as in FM. On the other hand, we can characterize more precisely this ratio using our proposed definitions:  and  (Eqs. 13, 14) based on the data from (A) and (B)
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC5037163&req=5

Fig1: Conceptual comparison of EM colocalization coefficients versus the overlap in FM. a The colocalization in EM is based on the single-particle colocalization resulting in colocalizing pairs of particles on the given distance range. On the contrary, FM colocalization is based on the overlapping signals in pixels from the different color channels. b The relative frequency distribution of the colocalizing/non-colocalizing particles/pixels of the model examples in (A). After combining the information about the colocalizing particles, we conclude the additive characteristics of EM colocalization versus nonadditive but union characteristics of FM colocalization. Additionally, the first two bars for labels A and B in the bar graph from EM image include the relations between proposed coefficients: and (Eqs. 11, 12). c The Manders’ overlap coefficients in FM are calculated based on the intersection of the signals (overlap) and the union of the overlapped plus non-overlapped pixels of the labels in (A). The Manders’ overlap coefficient M1 for the label A can be alternatively calculated as division of the proportions (4/32)/(24/32) = 4/24, that is, 12.5 %/75.0 % ≈ 16.7 %. Coefficient M2 for the label B is: (4/32)/(12/32) = 4/12, that is, 12.5 %/37.5 % ≈ 33.3 %. The EM relative aggregated colocalization coefficients for the label A and for the label B are calculated in the similar manner from the frequency distribution of the colocalizing and non-colocalizing particles of the corresponding type (Eq. 4). Also, the alternative calculation is based on the identical approach as in FM. On the other hand, we can characterize more precisely this ratio using our proposed definitions: and (Eqs. 13, 14) based on the data from (A) and (B)
Mentions: To describe the colocalization situation more detailed, we derive another important statistical coefficients, which includes the integration of the first and second level coefficients. By this combination, we receive the set of four summary measures, which includes the complex information about the proportions of colocalizing particles (, ) and non-colocalizing particles (, ) of the given labeling on the average image (Fig. 1):Fig. 1

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.