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Novel methods of automated quantification of gap junction distribution and interstitial collagen quantity from animal and human atrial tissue sections.

Yan J, Thomson JK, Wu X, Zhao W, Pollard AE, Ai X - PLoS ONE (2014)

Bottom Line: This approach allowed segmentation between ID-associated and non-ID-associated Cx43.Our results strongly demonstrate that the two novel image-processing approaches can minimize potential overestimation or underestimation of gap junction and structural remodeling in healthy and pathological hearts.The results of using the two novel methods will significantly improve our understanding of the molecular and structural remodeling associated functional changes in cardiac arrhythmia development in aged and diseased hearts.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell and Molecular Physiology, Loyola University Chicago, Maywood, Illinois, United States of America.

ABSTRACT

Background: Gap junctions (GJs) are the principal membrane structures that conduct electrical impulses between cardiac myocytes while interstitial collagen (IC) can physically separate adjacent myocytes and limit cell-cell communication. Emerging evidence suggests that both GJ and interstitial structural remodeling are linked to cardiac arrhythmia development. However, automated quantitative identification of GJ distribution and IC deposition from microscopic histological images has proven to be challenging. Such quantification is required to improve the understanding of functional consequences of GJ and structural remodeling in cardiac electrophysiology studies.

Methods and results: Separate approaches were employed for GJ and IC identification in images from histologically stained tissue sections obtained from rabbit and human atria. For GJ identification, we recognized N-Cadherin (N-Cad) as part of the gap junction connexin 43 (Cx43) molecular complex. Because N-Cad anchors Cx43 on intercalated discs (ID) to form functional GJ channels on cell membranes, we computationally dilated N-Cad pixels to create N-Cad units that covered all ID-associated Cx43 pixels on Cx43/N-Cad double immunostained confocal images. This approach allowed segmentation between ID-associated and non-ID-associated Cx43. Additionally, use of N-Cad as a unique internal reference with Z-stack layer-by-layer confocal images potentially limits sample processing related artifacts in Cx43 quantification. For IC quantification, color map thresholding of Masson's Trichrome blue stained sections allowed straightforward and automated segmentation of collagen from non-collagen pixels. Our results strongly demonstrate that the two novel image-processing approaches can minimize potential overestimation or underestimation of gap junction and structural remodeling in healthy and pathological hearts. The results of using the two novel methods will significantly improve our understanding of the molecular and structural remodeling associated functional changes in cardiac arrhythmia development in aged and diseased hearts.

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Confocal images of double immunofluorescence staining with Cx43 (green) and N-Cadherin (N-Cad; red) antibodies in rabbit left atrium (LA).A-C. Representative confocal images of Cx43/N-Cad overlay (A), N-Cad (red; B) and Cx43 (green; C). D-F. Enlarged images from a cropped area of image A including the overlay image (D), N-Cad pixel-by-pixel matched Cx43 (E), and N-Cad non-pixel-by-pixel Cx43 (F). Arrows indicate stellate Cx43 pixels that are in close proximity with the N-Cad pixel but do not co-localize with N-Cad pixel-by-pixel. G. Representative histogram of quantified pixel intensity of Cx43 stained image. H. Representative plot for the mean intensity (blue curve) and standard deviation (red curve) of foreground pixels at different thresholds. I. Representative plot of JT scores corresponding to R-value of four images from two young and two aged rabbit LA. J. Data plot of maximum JT scores of the images from four young and four aged rabbit LA.
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pone-0104357-g001: Confocal images of double immunofluorescence staining with Cx43 (green) and N-Cadherin (N-Cad; red) antibodies in rabbit left atrium (LA).A-C. Representative confocal images of Cx43/N-Cad overlay (A), N-Cad (red; B) and Cx43 (green; C). D-F. Enlarged images from a cropped area of image A including the overlay image (D), N-Cad pixel-by-pixel matched Cx43 (E), and N-Cad non-pixel-by-pixel Cx43 (F). Arrows indicate stellate Cx43 pixels that are in close proximity with the N-Cad pixel but do not co-localize with N-Cad pixel-by-pixel. G. Representative histogram of quantified pixel intensity of Cx43 stained image. H. Representative plot for the mean intensity (blue curve) and standard deviation (red curve) of foreground pixels at different thresholds. I. Representative plot of JT scores corresponding to R-value of four images from two young and two aged rabbit LA. J. Data plot of maximum JT scores of the images from four young and four aged rabbit LA.

Mentions: Images of Cx43 and N-Cad double immuno-stained atrial tissue sections were obtained using a Z-stack mode with a laser scanning confocal microscope (Zeiss; 40x magnification). Twenty to twenty five Z-stack confocal images with muscle fiber orientation parallel to the focal plane from each specimen were acquired with each Z-stack image file containing sequential images from 8–10 scanning layers. Each individual image of the Z-stack file was converted into 6–10 “tif” files at 8-bit data depth. A maximal projection image combining all the sequential images was also processed and saved using “tif” format. Each confocal fluorescent image “tif” file was encoded on three channels: red stained N-Cad channel (R), green stained Cx43 channel (G), and blue stained nuclei channel (B). Two grayscale images were converted from the R and G channels for quantitative image processing (Figs. 1A-1C) such that pixels with value 0 represented complete negative staining and pixels with value 255 represented complete saturation (Fig. 1G). According to a threshold method developed by Ostu N, [27] an ideal threshold that differentiates the foreground from the background and gives the minimized weighted sum of within-class variances can be determined by a maximal value of the JT score where JT = [P1*P2*(μ1-μ2)2]/(σ12+σ22). P1 and P2 are the number of pixels in the two groups (foreground and background), μ1 and μ2 are the mean intensity level of the two groups, and σ1 and σ2 are their standard deviations. For each image, we found that mean intensity of selected foreground pixels was increased along with incrementally changed thresholds, meanwhile the value of the standard deviation was initially increased and then decreased after a maximal peak was reached (Fig. 1H). Thus, the initial threshold for positively stained Cx43 pixels was selected based on the maximal standard deviation (σgreen) on each image (Fig. 1H). A simplified formula: µgreen+R* σgreen was then applied in our algorithm for each individual image to define an optimal threshold (where R is a variable that gives a maximal JT score and µgreen is the mean of selected Cx43 pixels at the initial threshold). To determine optimal Rs for Cx43 immunostained images from young and aged rabbit atrial sections (n = 4, 4), JT values of a total of 108 images with Rs ranging from −3 to 3 were calculated. We found that the maximum JT values of the images from both young and aged rabbit atrial sections were very close to an R-value of −1.18 (μYg = −1.12, σYg = 0.027, µAged = −1.24, σAged = 0.021; Figs. 1I & 1J). Positively stained Cx43 pixels were identified based on the optimal R-value. Finally, the sum of intensities of all positively stained pixels from each image was calculated as the quantified result.


Novel methods of automated quantification of gap junction distribution and interstitial collagen quantity from animal and human atrial tissue sections.

Yan J, Thomson JK, Wu X, Zhao W, Pollard AE, Ai X - PLoS ONE (2014)

Confocal images of double immunofluorescence staining with Cx43 (green) and N-Cadherin (N-Cad; red) antibodies in rabbit left atrium (LA).A-C. Representative confocal images of Cx43/N-Cad overlay (A), N-Cad (red; B) and Cx43 (green; C). D-F. Enlarged images from a cropped area of image A including the overlay image (D), N-Cad pixel-by-pixel matched Cx43 (E), and N-Cad non-pixel-by-pixel Cx43 (F). Arrows indicate stellate Cx43 pixels that are in close proximity with the N-Cad pixel but do not co-localize with N-Cad pixel-by-pixel. G. Representative histogram of quantified pixel intensity of Cx43 stained image. H. Representative plot for the mean intensity (blue curve) and standard deviation (red curve) of foreground pixels at different thresholds. I. Representative plot of JT scores corresponding to R-value of four images from two young and two aged rabbit LA. J. Data plot of maximum JT scores of the images from four young and four aged rabbit LA.
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Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4126721&req=5

pone-0104357-g001: Confocal images of double immunofluorescence staining with Cx43 (green) and N-Cadherin (N-Cad; red) antibodies in rabbit left atrium (LA).A-C. Representative confocal images of Cx43/N-Cad overlay (A), N-Cad (red; B) and Cx43 (green; C). D-F. Enlarged images from a cropped area of image A including the overlay image (D), N-Cad pixel-by-pixel matched Cx43 (E), and N-Cad non-pixel-by-pixel Cx43 (F). Arrows indicate stellate Cx43 pixels that are in close proximity with the N-Cad pixel but do not co-localize with N-Cad pixel-by-pixel. G. Representative histogram of quantified pixel intensity of Cx43 stained image. H. Representative plot for the mean intensity (blue curve) and standard deviation (red curve) of foreground pixels at different thresholds. I. Representative plot of JT scores corresponding to R-value of four images from two young and two aged rabbit LA. J. Data plot of maximum JT scores of the images from four young and four aged rabbit LA.
Mentions: Images of Cx43 and N-Cad double immuno-stained atrial tissue sections were obtained using a Z-stack mode with a laser scanning confocal microscope (Zeiss; 40x magnification). Twenty to twenty five Z-stack confocal images with muscle fiber orientation parallel to the focal plane from each specimen were acquired with each Z-stack image file containing sequential images from 8–10 scanning layers. Each individual image of the Z-stack file was converted into 6–10 “tif” files at 8-bit data depth. A maximal projection image combining all the sequential images was also processed and saved using “tif” format. Each confocal fluorescent image “tif” file was encoded on three channels: red stained N-Cad channel (R), green stained Cx43 channel (G), and blue stained nuclei channel (B). Two grayscale images were converted from the R and G channels for quantitative image processing (Figs. 1A-1C) such that pixels with value 0 represented complete negative staining and pixels with value 255 represented complete saturation (Fig. 1G). According to a threshold method developed by Ostu N, [27] an ideal threshold that differentiates the foreground from the background and gives the minimized weighted sum of within-class variances can be determined by a maximal value of the JT score where JT = [P1*P2*(μ1-μ2)2]/(σ12+σ22). P1 and P2 are the number of pixels in the two groups (foreground and background), μ1 and μ2 are the mean intensity level of the two groups, and σ1 and σ2 are their standard deviations. For each image, we found that mean intensity of selected foreground pixels was increased along with incrementally changed thresholds, meanwhile the value of the standard deviation was initially increased and then decreased after a maximal peak was reached (Fig. 1H). Thus, the initial threshold for positively stained Cx43 pixels was selected based on the maximal standard deviation (σgreen) on each image (Fig. 1H). A simplified formula: µgreen+R* σgreen was then applied in our algorithm for each individual image to define an optimal threshold (where R is a variable that gives a maximal JT score and µgreen is the mean of selected Cx43 pixels at the initial threshold). To determine optimal Rs for Cx43 immunostained images from young and aged rabbit atrial sections (n = 4, 4), JT values of a total of 108 images with Rs ranging from −3 to 3 were calculated. We found that the maximum JT values of the images from both young and aged rabbit atrial sections were very close to an R-value of −1.18 (μYg = −1.12, σYg = 0.027, µAged = −1.24, σAged = 0.021; Figs. 1I & 1J). Positively stained Cx43 pixels were identified based on the optimal R-value. Finally, the sum of intensities of all positively stained pixels from each image was calculated as the quantified result.

Bottom Line: This approach allowed segmentation between ID-associated and non-ID-associated Cx43.Our results strongly demonstrate that the two novel image-processing approaches can minimize potential overestimation or underestimation of gap junction and structural remodeling in healthy and pathological hearts.The results of using the two novel methods will significantly improve our understanding of the molecular and structural remodeling associated functional changes in cardiac arrhythmia development in aged and diseased hearts.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell and Molecular Physiology, Loyola University Chicago, Maywood, Illinois, United States of America.

ABSTRACT

Background: Gap junctions (GJs) are the principal membrane structures that conduct electrical impulses between cardiac myocytes while interstitial collagen (IC) can physically separate adjacent myocytes and limit cell-cell communication. Emerging evidence suggests that both GJ and interstitial structural remodeling are linked to cardiac arrhythmia development. However, automated quantitative identification of GJ distribution and IC deposition from microscopic histological images has proven to be challenging. Such quantification is required to improve the understanding of functional consequences of GJ and structural remodeling in cardiac electrophysiology studies.

Methods and results: Separate approaches were employed for GJ and IC identification in images from histologically stained tissue sections obtained from rabbit and human atria. For GJ identification, we recognized N-Cadherin (N-Cad) as part of the gap junction connexin 43 (Cx43) molecular complex. Because N-Cad anchors Cx43 on intercalated discs (ID) to form functional GJ channels on cell membranes, we computationally dilated N-Cad pixels to create N-Cad units that covered all ID-associated Cx43 pixels on Cx43/N-Cad double immunostained confocal images. This approach allowed segmentation between ID-associated and non-ID-associated Cx43. Additionally, use of N-Cad as a unique internal reference with Z-stack layer-by-layer confocal images potentially limits sample processing related artifacts in Cx43 quantification. For IC quantification, color map thresholding of Masson's Trichrome blue stained sections allowed straightforward and automated segmentation of collagen from non-collagen pixels. Our results strongly demonstrate that the two novel image-processing approaches can minimize potential overestimation or underestimation of gap junction and structural remodeling in healthy and pathological hearts. The results of using the two novel methods will significantly improve our understanding of the molecular and structural remodeling associated functional changes in cardiac arrhythmia development in aged and diseased hearts.

Show MeSH
Related in: MedlinePlus