Automatic Quantification of Cardiomyocyte Dimensions and Connexin 43 Lateralization in Fluorescence Images
<p>Input fluorescence microscopy images. <span class="html-italic">A</span>, <span class="html-italic">B</span>, <span class="html-italic">C</span>, from left to right: human samples from three individuals <span class="html-italic">a</span>, <span class="html-italic">b</span> and <span class="html-italic">c</span>. <span class="html-italic">D</span> and <span class="html-italic">E</span>, from left to right: rat samples <math display="inline"><semantics> <mrow> <mi>d</mi> <mn>2</mn> </mrow> </semantics></math> and <span class="html-italic">e</span>. Green signal corresponds to sarco/endoplasmic reticulum <math display="inline"><semantics> <mrow> <mi>C</mi> <msup> <mi>a</mi> <mrow> <mn>2</mn> <mo>+</mo> </mrow> </msup> </mrow> </semantics></math> ATPase (SERCA2) in humans and F-actin in rat samples (channel <math display="inline"><semantics> <msub> <mi>c</mi> <mn>1</mn> </msub> </semantics></math>). White signal marks connexin 43 (CX43) (channel <math display="inline"><semantics> <msub> <mi>c</mi> <mn>2</mn> </msub> </semantics></math>). Red signal is wheat germ agglutinin (WGA) in human samples (channel <math display="inline"><semantics> <msub> <mi>c</mi> <mn>3</mn> </msub> </semantics></math>). Scale bar is in all cases 100 <math display="inline"><semantics> <mi mathvariant="sans-serif">μ</mi> </semantics></math>m.</p> "> Figure 2
<p>Image of a left ventricular rat tissue partly analyzed in this study (<b>left</b>) and three insets at higher magnification (<b>right</b>). Green signal represents F-actin (channel <math display="inline"><semantics> <msub> <mi>c</mi> <mn>1</mn> </msub> </semantics></math>). White signal is CX43 (channel <math display="inline"><semantics> <msub> <mi>c</mi> <mn>2</mn> </msub> </semantics></math>). Within the myocardium, black background estimates the interstitium (channel <math display="inline"><semantics> <msub> <mi>c</mi> <mn>3</mn> </msub> </semantics></math>). Scale bar: left, 1000 <math display="inline"><semantics> <mi mathvariant="sans-serif">μ</mi> </semantics></math>m; right insets, 100 <math display="inline"><semantics> <mi mathvariant="sans-serif">μ</mi> </semantics></math>m.</p> "> Figure 3
<p>Left panel: representation of vertex projection from original coordinates (<math display="inline"><semantics> <mrow> <msub> <mi>x</mi> <mrow> <mi>v</mi> <mo>,</mo> <mi>i</mi> </mrow> </msub> <mo>,</mo> <msub> <mi>y</mi> <mrow> <mi>v</mi> <mo>,</mo> <mi>i</mi> </mrow> </msub> </mrow> </semantics></math>) to extended coordinates (<math display="inline"><semantics> <mrow> <msubsup> <mi>x</mi> <mrow> <mi>v</mi> <mo>,</mo> <mi>i</mi> </mrow> <mo>*</mo> </msubsup> <mo>,</mo> <msubsup> <mi>y</mi> <mrow> <mi>v</mi> <mo>,</mo> <mi>i</mi> </mrow> <mo>*</mo> </msubsup> </mrow> </semantics></math>). Right panel: illustration of how the intersection between cardiomyocytes (CMs) in the manual and automatic masks was computed. The red box <math display="inline"><semantics> <msub> <mi>B</mi> <msub> <mi>a</mi> <mi>j</mi> </msub> </msub> </semantics></math> shows the enclosing rectangle for a CM in the automatic mask <math display="inline"><semantics> <msub> <mi>M</mi> <mi>a</mi> </msub> </semantics></math>, whereas the blue box <math display="inline"><semantics> <msub> <mi>B</mi> <msub> <mi>m</mi> <mi>i</mi> </msub> </msub> </semantics></math> shows the enclosing rectangle for a CM in the manual mask <math display="inline"><semantics> <msub> <mi>M</mi> <mi>m</mi> </msub> </semantics></math>. The purple area is the intersection between the areas of the two rectangles: <math display="inline"><semantics> <mrow> <msub> <mi>A</mi> <msub> <mi>a</mi> <mi>j</mi> </msub> </msub> <mspace width="2.0pt"/> <mspace width="2.0pt"/> <mo>∩</mo> <msub> <mi>A</mi> <msub> <mi>m</mi> <mi>i</mi> </msub> </msub> </mrow> </semantics></math>.</p> "> Figure 4
<p>Top: <math display="inline"><semantics> <msub> <mi>M</mi> <mrow> <mi>c</mi> <mn>1</mn> </mrow> </msub> </semantics></math> (SERCA2) and <math display="inline"><semantics> <msub> <mi>M</mi> <mrow> <mi>c</mi> <mn>3</mn> </mrow> </msub> </semantics></math> (WGA) binarized channels. Bottom left: <math display="inline"><semantics> <msub> <mi>M</mi> <mrow> <mi>c</mi> <mn>2</mn> </mrow> </msub> </semantics></math> (CX43) binarized channel. Bottom right: merged channel. Scale bar = 100 <math display="inline"><semantics> <mi mathvariant="sans-serif">μ</mi> </semantics></math>m.</p> "> Figure 5
<p>Merged channels for image <span class="html-italic">a</span> before (<b>left</b>) and after (<b>right</b>) noise removal. Scale bar = 100 <math display="inline"><semantics> <mi mathvariant="sans-serif">μ</mi> </semantics></math>m.</p> "> Figure 6
<p>Manual versus automatic cell delineation in image <span class="html-italic">a</span>. Top left: mask <math display="inline"><semantics> <msub> <mi>M</mi> <mi>m</mi> </msub> </semantics></math> obtained by manual delineation of CMs’ boundaries in image <span class="html-italic">a</span>. Top right: mask <math display="inline"><semantics> <msub> <mi>M</mi> <mi>a</mi> </msub> </semantics></math> generated by Myocyte Automatic Retrieval and Tissue Analyzer (MARTA) software for image <span class="html-italic">a</span>. Bottom left: CMs’ contours for manual (white) and automatic (green) masks and corresponding enclosing rectangles (blue for manual, red for automatic). Bottom right: Enclosing rectangles from manual and automatic masks showing overlapping above 50%. Scale bar = 100 <math display="inline"><semantics> <mi mathvariant="sans-serif">μ</mi> </semantics></math>m.</p> "> Figure 7
<p>Percentile curves of maximum intersection <math display="inline"><semantics> <msub> <mi>I</mi> <mi>i</mi> </msub> </semantics></math> for CMs <span class="html-italic">i</span> in <math display="inline"><semantics> <msub> <mi>M</mi> <mi>m</mi> </msub> </semantics></math>, for all the analyzed images. Area Under the Curve (AUC) values are presented in the legend.</p> "> Figure 8
<p>CMs in the manual (<b>left</b>) and automatic (<b>right</b>) masks contoured in pink, with enclosing rectangles in yellow. At the bottom right corner of each panel is an example of morphological measures computed for a CM detected in both masks. Scale bar = 100 <math display="inline"><semantics> <mi mathvariant="sans-serif">μ</mi> </semantics></math>m.</p> "> Figure 9
<p>Box plots of length <span class="html-italic">L</span> (top left), width <span class="html-italic">W</span> (top right), length-to-width ratio <span class="html-italic">R</span> (bottom left) and area <span class="html-italic">A</span> (bottom right) for manual (in blue) and automatic (in red) masks, calculated for images <span class="html-italic">a</span>, <span class="html-italic">b</span> and <span class="html-italic">c</span>.</p> "> Figure 10
<p>Left: Tissue mask <math display="inline"><semantics> <msub> <mi>M</mi> <mi>t</mi> </msub> </semantics></math> represented in white, enclosed by maximum contour in red, for image <span class="html-italic">a</span>. Right: Equalized and binarized channel <math display="inline"><semantics> <msub> <mi>M</mi> <mrow> <mi>c</mi> <mn>4</mn> </mrow> </msub> </semantics></math> (<math display="inline"><semantics> <mrow> <mi>t</mi> <mi>h</mi> <msub> <mi>r</mi> <mrow> <mi>c</mi> <mn>4</mn> </mrow> </msub> <mo>=</mo> <mn>254</mn> </mrow> </semantics></math>) for the same image. Scale bar = 100 <math display="inline"><semantics> <mi mathvariant="sans-serif">μ</mi> </semantics></math>m.</p> "> Figure 11
<p>Representative images of box partitioning for two CMs A and B of the manual <math display="inline"><semantics> <msub> <mi>M</mi> <mi>m</mi> </msub> </semantics></math> (<b>left</b>) and automatic <math display="inline"><semantics> <msub> <mi>M</mi> <mi>a</mi> </msub> </semantics></math> (<b>right</b>) masks. Top: overlay of channels <math display="inline"><semantics> <msub> <mi>c</mi> <mn>1</mn> </msub> </semantics></math> (SERCA2, green), <math display="inline"><semantics> <msub> <mi>c</mi> <mn>2</mn> </msub> </semantics></math> (CX43, white) and <math display="inline"><semantics> <msub> <mi>c</mi> <mn>3</mn> </msub> </semantics></math> (WGA, red) and results of CMs contour in pink, and the minimum area rectangles enclosing them in yellow. Rectangles are divided into four regions corresponding to polar (i.e., end-to-end) and lateral (i.e., middle) cell compartments. Bottom: channel <math display="inline"><semantics> <msub> <mi>c</mi> <mn>2</mn> </msub> </semantics></math> without binarization. Polar CX43 bands appear as discontinuous line patterns because of the low signal intensity. Scale bar = 20 <math display="inline"><semantics> <mi mathvariant="sans-serif">μ</mi> </semantics></math>m.</p> "> Figure 12
<p>Histograms and density plots for <math display="inline"><semantics> <msub> <mi>C</mi> <mrow> <mi>l</mi> <mi>a</mi> <mi>t</mi> </mrow> </msub> </semantics></math> computed for images <span class="html-italic">a</span> (<b>top left</b>), <span class="html-italic">b</span> (<b>top right</b>) and <span class="html-italic">c</span> (<b>bottom left</b>), as well as for pooled data from the three images (<b>bottom right</b>), both from automatic (red) and manual (blue) CM masks.</p> "> Figure 13
<p>Detected CMs in the automatic mask <math display="inline"><semantics> <msub> <mi>M</mi> <mi>a</mi> </msub> </semantics></math> computed under the supervised mode for images <math display="inline"><semantics> <mrow> <mi>d</mi> <mn>1</mn> </mrow> </semantics></math> (<b>left</b>) and <math display="inline"><semantics> <mrow> <mi>d</mi> <mn>2</mn> </mrow> </semantics></math> (<b>right</b>). Scale bar = 100 <math display="inline"><semantics> <mi mathvariant="sans-serif">μ</mi> </semantics></math>m.</p> "> Figure 14
<p>Examples of four detected CMs from automatic masks obtained for images <math display="inline"><semantics> <mrow> <mi>d</mi> <mn>1</mn> </mrow> </semantics></math> (<b>top</b>) and <math display="inline"><semantics> <mrow> <mi>d</mi> <mn>2</mn> </mrow> </semantics></math> (<b>bottom</b>). Morphological measurements and <math display="inline"><semantics> <msub> <mi>C</mi> <mrow> <mi>l</mi> <mi>a</mi> <mi>t</mi> </mrow> </msub> </semantics></math> values are provided in <a href="#biomolecules-10-01334-t003" class="html-table">Table 3</a> for <math display="inline"><semantics> <mrow> <mi>d</mi> <msub> <mn>1</mn> <mrow> <mi>C</mi> <mi>M</mi> <mo>,</mo> <mn>1</mn> </mrow> </msub> </mrow> </semantics></math> (<b>top left</b>), <math display="inline"><semantics> <mrow> <mi>d</mi> <msub> <mn>1</mn> <mrow> <mi>C</mi> <mi>M</mi> <mo>,</mo> <mn>2</mn> </mrow> </msub> </mrow> </semantics></math> (<b>top right</b>), <math display="inline"><semantics> <mrow> <mi>d</mi> <msub> <mn>2</mn> <mrow> <mi>C</mi> <mi>M</mi> <mo>,</mo> <mn>3</mn> </mrow> </msub> </mrow> </semantics></math> (<b>bottom left</b>) and <math display="inline"><semantics> <mrow> <mi>d</mi> <msub> <mn>2</mn> <mrow> <mi>C</mi> <mi>M</mi> <mo>,</mo> <mn>4</mn> </mrow> </msub> </mrow> </semantics></math> (<b>bottom right</b>). Scale bar = 20 <math display="inline"><semantics> <mi mathvariant="sans-serif">μ</mi> </semantics></math>m.</p> "> Figure 15
<p>Binarized channels representing the CM mask <math display="inline"><semantics> <msub> <mi>M</mi> <mrow> <mi>c</mi> <mn>1</mn> </mrow> </msub> </semantics></math> (<b>top left</b>, in green), the interstitium generated by inverting the CM mask (<b>top right</b>, in red) and the CX43 mask <math display="inline"><semantics> <msub> <mi>M</mi> <mrow> <mi>c</mi> <mn>2</mn> </mrow> </msub> </semantics></math> (<b>bottom left</b>, in white), as well as original image <span class="html-italic">e</span> with overlaid CMs detected in supervised mode (<b>bottom right</b>). Scale bar = 100 <math display="inline"><semantics> <mi mathvariant="sans-serif">μ</mi> </semantics></math>m.</p> "> Figure 16
<p>Four detected CMs from the automatic mask <math display="inline"><semantics> <msub> <mi>M</mi> <mi>a</mi> </msub> </semantics></math> on top of image <span class="html-italic">e</span>. Morphological measurements and <math display="inline"><semantics> <msub> <mi>C</mi> <mrow> <mi>l</mi> <mi>a</mi> <mi>t</mi> </mrow> </msub> </semantics></math> values are provided in <a href="#biomolecules-10-01334-t003" class="html-table">Table 3</a> for <math display="inline"><semantics> <msub> <mi>e</mi> <mrow> <mi>C</mi> <mi>M</mi> <mo>,</mo> <mn>1</mn> </mrow> </msub> </semantics></math> (<b>top left</b>), <math display="inline"><semantics> <msub> <mi>e</mi> <mrow> <mi>C</mi> <mi>M</mi> <mo>,</mo> <mn>2</mn> </mrow> </msub> </semantics></math> (<b>top right</b>), <math display="inline"><semantics> <msub> <mi>e</mi> <mrow> <mi>C</mi> <mi>M</mi> <mo>,</mo> <mn>3</mn> </mrow> </msub> </semantics></math> (<b>bottom left</b>) and <math display="inline"><semantics> <msub> <mi>e</mi> <mrow> <mi>C</mi> <mi>M</mi> <mo>,</mo> <mn>4</mn> </mrow> </msub> </semantics></math> (<b>bottom right</b>). Scale bar = 20 <math display="inline"><semantics> <mi mathvariant="sans-serif">μ</mi> </semantics></math>m.</p> ">
Abstract
:1. Introduction
2. Materials and Methods
2.1. Sample Collection and Fluorescent Immunohistochemistry
2.2. From Fluorescence Images to Cardiomyocyte Binary Masks
2.3. Cardiomyocyte Detection and Morphological Characterization
2.4. Quantification of CX43 Expression
2.5. Quantification of CX43 Distribution
2.6. Performance Evaluation
3. Results
3.1. Automated Image Analysis
3.2. Agreement between Automatic and Manual Cell Delineation
3.3. Cardiomyocytes’ Morphological Measurements
3.4. Quantification of CX43 Expression
3.5. Determination of CX43 Distribution
3.6. Application to Images with One or Two Channels
3.7. Processing Time
4. Discussion
4.1. Cardiomyocytes’ Morphological Measurements
4.2. CX43 Expression and Distribution
4.3. Additional Features of the Proposed Software
4.4. Study Limitations and Future Extensions
Supplementary Materials
Author Contributions
Funding
Conflicts of Interest
Abbreviations
CX43 | Connexin43 |
CM | Cardiomyocite |
WGA | Wheat Germ Agglutinin |
LV | Left Ventricle |
SL | Sarcomere Length |
SERCA | Sarco/endoplasmic reticulum ATPase |
DsRed | Red Fluorescent Protein |
AUC | Area Under The Curve |
FITC | Fluorescein isothiocyanate |
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ID | a | b | c | e | ||
---|---|---|---|---|---|---|
♯ inputs | 3 | 3 | 3 | 1 | 1 | 1 |
♯ channels | 3 | 3 | 3 | 1 | 2 | 2 |
equalized (y/n) | n | n | n | y | y | y |
supervised (y/n) | n | n | n | y | y | y |
scale | 0.21 | 0.21 | 0.21 | 0.227 | 0.227 | 0.114 |
8 | 8 | 8 | 128 | 100 | 70 | |
15 | 15 | 15 | 254 | 254 | 254 | |
2 | 2 | 2 | 128 | 100 | 70 | |
254 | 254 | 254 | 254 | 254 | 254 | |
3 | 3 | 3 | 1 | 1 | 1 | |
3 | 3 | 3 | 3 | 3 | 2 | |
3 | 3 | 3 | 3 | 3 | 1 | |
3 | 3 | 3 | 1 | 1 | 1 | |
3 | 3 | 3 | 4 | 4 | 5 | |
5 | 5 | 5 | 8 | 8 | 20 | |
3 | 3 | 3 | 1 | 1 | 1 | |
3 | 3 | 3 | 3 | 3 | 2 | |
3 | 3 | 3 | 3 | 3 | 1 | |
66 | 453 | 64 | 111 (28) | 170 (44) | 15 (7) | |
84 | 371 | 82 | 344 | 344 | 46 |
(%) | (%) | (%) | (%) | (%) | (%) | |||
---|---|---|---|---|---|---|---|---|
252 | 1.41 | 0.111 | 1.53 | 0.063 | 2.63 | 0.353 | 0.54 | 0.58 |
253 | 0.82 | 0.039 | 0.97 | 0.031 | 1.60 | 0.169 | 0.51 | 0.61 |
254 | 0.27 | 0.007 | 0.31 | 0.007 | 0.58 | 0.039 | 0.47 | 0.53 |
CM | L (m) | W (m) | R | A (m) | (%) |
---|---|---|---|---|---|
166.9 | 44.5 | 3.7 | 7428.6 | 48.8 | |
120.3 | 25.2 | 4.8 | 3031.7 | 60.2 | |
107.7 | 35.1 | 3.1 | 3777.3 | 5.4 | |
108.2 | 25.2 | 4.3 | 2730.6 | 0.0 | |
81.6 | 35.6 | 2.3 | 2903.8 | 44.1 | |
111.1 | 33.3 | 3.3 | 3695.3 | 41.6 | |
42.7 | 17.9 | 2.4 | 762.9 | 0.0 | |
71.1 | 33.8 | 2.1 | 2399.0 | 11.9 |
ID | (s) | Ratio | (s) | S (Mb) | |||
---|---|---|---|---|---|---|---|
a | 0.11 | 1.0 | 66.0 | 600 | 22 | 66 | 52.0 |
b | 0.11 | 15.4 | 29.4 | 267 | 1036 | 453 | 363.0 |
c | 0.11 | 3.4 | 18.8 | 171 | 54 | 64 | 92.5 |
d1 | 0.11 | 4.6 | 24.1 (6.1) | 219 (55) | 201 (522) | 111 (28) | 18.3 |
d2 | 0.11 | 4.6 | 36.9 (9.6) | 335 (87) | 300 (683) | 170 (44) | 43.5 |
e | 0.11 | 2.1 | 7.1 (3.3) | 64 (30) | 26 (71) | 15 (7) | 18.7 |
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Oliver-Gelabert, A.; García-Mendívil, L.; Vallejo-Gil, J.M.; Fresneda-Roldán, P.C.; Andelová, K.; Fañanás-Mastral, J.; Vázquez-Sancho, M.; Matamala-Adell, M.; Sorribas-Berjón, F.; Ballester-Cuenca, C.; et al. Automatic Quantification of Cardiomyocyte Dimensions and Connexin 43 Lateralization in Fluorescence Images. Biomolecules 2020, 10, 1334. https://doi.org/10.3390/biom10091334
Oliver-Gelabert A, García-Mendívil L, Vallejo-Gil JM, Fresneda-Roldán PC, Andelová K, Fañanás-Mastral J, Vázquez-Sancho M, Matamala-Adell M, Sorribas-Berjón F, Ballester-Cuenca C, et al. Automatic Quantification of Cardiomyocyte Dimensions and Connexin 43 Lateralization in Fluorescence Images. Biomolecules. 2020; 10(9):1334. https://doi.org/10.3390/biom10091334
Chicago/Turabian StyleOliver-Gelabert, Antoni, Laura García-Mendívil, José María Vallejo-Gil, Pedro Carlos Fresneda-Roldán, Katarína Andelová, Javier Fañanás-Mastral, Manuel Vázquez-Sancho, Marta Matamala-Adell, Fernando Sorribas-Berjón, Carlos Ballester-Cuenca, and et al. 2020. "Automatic Quantification of Cardiomyocyte Dimensions and Connexin 43 Lateralization in Fluorescence Images" Biomolecules 10, no. 9: 1334. https://doi.org/10.3390/biom10091334