3D-Reconstructed Retinal Pigment Epithelial Cells Provide Insights into the Anatomy of the Outer Retina
Abstract
:1. Introduction
2. Results
3. Discussion
4. Materials and Methods
4.1. Animal Procedures
4.2. Preparation of Flatmounts
4.3. Preparation of Eyes for SBF-SEM
4.4. SBF-SEM
4.5. Segmentation and 3D Reconstructions
4.6. Analysis
object1 = “Untitled”; |
object2 = “Untitled”; |
Dialog.create(“Measuring shared area between”); |
Dialog.addString(“Object1:”, object1); |
Dialog.addString(“Object2:”, object2); |
Dialog.show(); |
object1 = Dialog.getString(); |
object2 = Dialog.getString(); |
path = File.directory(); |
title_orig = getTitle(); |
getVoxelSize(width, height, depth, unit); |
pixelWidth = width; |
pixelDepth = depth; |
pixelUnit = unit; |
junctionArea_total = 0; |
junctionArea_array = newArray(0); |
imageNbr = nSlices(); |
for(i = 1; i < imageNbr; i++){ |
selectWindow(title_orig); |
setSlice(i); |
run(“Duplicate...”, “title = Object1.tif”); |
setAutoThreshold(“Default”); |
//run(“Threshold...”); |
setAutoThreshold(“Default dark”); |
setThreshold(1, 1); |
setOption(“BlackBackground”, false); |
run(“Convert to Mask”); |
rename(“Object1_binary.tif”); |
run(“Dilate”); |
selectWindow(title_orig); |
run(“Duplicate...”, “title = Object2.tif”); |
setAutoThreshold(“Default”); |
//run(“Threshold...”); |
setAutoThreshold(“Default dark”); |
setThreshold(2, 2); |
setOption(“BlackBackground”, false); |
run(“Convert to Mask”); |
rename(“Object2_binary.tif”); |
//run(“Dilate”); |
//run(“Fill Holes”); |
imageCalculator(“AND create”, “Object1_binary.tif”,”Object2_binary.tif”); |
selectWindow(“Object1_binary.tif”); |
close(); |
selectWindow(“Object2_binary.tif”); |
close(); |
selectWindow(“Result of Object1_binary.tif”); |
setAutoThreshold(“Default dark”); |
//run(“Threshold...”); |
setAutoThreshold(“Default”); |
run(“Create Selection”); |
getSelectionBounds(xRect0, yRect0, xWidth, yHeight); |
selectionArray = newArray(xWidth*yHeight); |
a = 0; |
for(y = yRect0; y < yRect0 + yHeight; y++){ |
for(x = xRect0; x < xRect0 + xWidth; x++){ |
selectionArray[a++] = getPixel(x,y); |
} |
} |
selectionArray2 = newArray(0); |
for(c = 0; c < selectionArray.length; c++){ |
if (selectionArray[c] > 254){ |
selectionArray2 = Array.concat(selectionArray2, selectionArray[c]); |
} |
} |
nbrPixel = selectionArray2.length; |
junctionLength = nbrPixel * pixelWidth; |
junctionArea = junctionLength * pixelDepth; |
junctionArea_array = Array.concat(junctionArea_array, junctionArea); |
junctionArea_total = junctionArea_total + junctionArea; |
selectWindow(“Result of Object1_binary.tif”); |
close(); |
} |
if (nResults > =0) { |
run(“Clear Results”); |
} |
i = nResults; |
for (n = 0; n < junctionArea_array.length; n++){ |
setResult(“Slice”, i, n); |
setResult(“Pixel_Unit”, 0, pixelUnit); |
setResult(“Surf.Area_stack”, 0, junctionArea_total); |
setResult(“Surf.Area_slices”, i, junctionArea_array[n]); |
i = nResults; |
} |
updateResults; |
saveAs(“Results”, path + “SharedSA_”+object1+”_”+object2+”.csv”); |
run(“Close”); |
selectWindow(title_orig); |
close(); |
exit(“ANALYSIS FINISHED”); |
4.7. Statistical Analysis
Supplementary Materials
Author Contributions
Funding
Acknowledgments
Conflicts of Interest
References
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Cell 1 | Cell 2 | Cell 3 | Cell 4 | Cell 5 | ||||||
---|---|---|---|---|---|---|---|---|---|---|
Mono-Nucleate (Whole Cell) | Bi-Nucleate (Whole Cell) | Bi-Nucleate (Partial Cell) | Bi-Nucleate (Partial Cell) | Bi-Nucleate (Whole Cell) | ||||||
Cell cytoplasm | Volume (µm3) | 2220.2 | 2360 | 1733 | 1803 | 2649 | ||||
Surface area (µm2) | 1794.9 | 2104 | 1572 | 1754.7 | 2055.6 | |||||
Microvilli | Volume (µm3) | 528.5 | 877 | 60.9 | 181 | 1158 | ||||
Surface area (µm2) | 1556.4 | 2617.7 | 1814.5 | 3165.2 | 3146.3 | |||||
Nuclei | Volume (µm3) | 141 | 146.8 | 144 | 139.5 | 126.5 | 128.2 | 138.1 | 123.6 | 131.6 |
Surface area (µm2) | 172.1 | 187.3 | 187 | 171.5 | 158.1 | 164.2 | 169.2 | 156.3 | 156.7 | |
Basal infolds (sub-RPE spaces) | Volume (µm3) | 40.9 | 164.1 | 92.4 | 116.7 | 172 | ||||
Surface area (µm2) | 537.9 | 1637.1 | 1682.2 | 1884.3 | 2505 | |||||
Surface of RPE microvilli in contact with photoreceptors (?m2) | 51,462 | 90,000 | 48,732 | 73,118 | 242,797 | |||||
Number of photoreceptors supported per volume (?m3) of RPE cytoplasm | 0.041 | 0.059 | 0.059 | 0.12 | 0.041 | |||||
Number of photoreceptors supported | 90 | 132 | 102 | 216 | 108 |
Mono-Nucleate RPE Cell | Bi-Nucleate RPE Cell | |
---|---|---|
(Cell 1) | (Cell 2) | |
Number of mitochondria | 422 | 678 |
Average mitochondrial volume (nm3) | 2.76 × 108 ± 4.38 × 108 SD | 3.04 × 108 ± 3.87 × 108 SD |
Volume of the smallest mitochondria (nm3) | 2.35 × 104 | 2.69 × 104 |
Total mitochondrial volume (nm3) | 1.17 × 1011 | 4.36 × 1011 |
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Keeling, E.; Chatelet, D.S.; Tan, N.Y.T.; Khan, F.; Richards, R.; Thisainathan, T.; Goggin, P.; Page, A.; Tumbarello, D.A.; Lotery, A.J.; et al. 3D-Reconstructed Retinal Pigment Epithelial Cells Provide Insights into the Anatomy of the Outer Retina. Int. J. Mol. Sci. 2020, 21, 8408. https://doi.org/10.3390/ijms21218408
Keeling E, Chatelet DS, Tan NYT, Khan F, Richards R, Thisainathan T, Goggin P, Page A, Tumbarello DA, Lotery AJ, et al. 3D-Reconstructed Retinal Pigment Epithelial Cells Provide Insights into the Anatomy of the Outer Retina. International Journal of Molecular Sciences. 2020; 21(21):8408. https://doi.org/10.3390/ijms21218408
Chicago/Turabian StyleKeeling, Eloise, David S. Chatelet, Nicole Y. T. Tan, Farihah Khan, Rhys Richards, Thibana Thisainathan, Patricia Goggin, Anton Page, David A. Tumbarello, Andrew J. Lotery, and et al. 2020. "3D-Reconstructed Retinal Pigment Epithelial Cells Provide Insights into the Anatomy of the Outer Retina" International Journal of Molecular Sciences 21, no. 21: 8408. https://doi.org/10.3390/ijms21218408
APA StyleKeeling, E., Chatelet, D. S., Tan, N. Y. T., Khan, F., Richards, R., Thisainathan, T., Goggin, P., Page, A., Tumbarello, D. A., Lotery, A. J., & Ratnayaka, J. A. (2020). 3D-Reconstructed Retinal Pigment Epithelial Cells Provide Insights into the Anatomy of the Outer Retina. International Journal of Molecular Sciences, 21(21), 8408. https://doi.org/10.3390/ijms21218408