Journal: Cells

Article Title: Extracellular Matrix Structure and Composition in the Early Four-Chambered Embryonic Heart

doi: 10.3390/cells9020285

Figure Lengend Snippet: Schematic of the process to image and analyze the embryonic myocardium at the microscale and macroscale. ( A ) Chick embryonic hearts were dissected, fixed, and stained at different stages of development. ( B ) To study the extracellular matrix (ECM) at the microscale, the left ventricle was dissected and opened to lay flat. ( C – E , i) High-resolution 3D images of the fibronectin, laminin, and collagen type IV ECM were obtained by confocal microscopy. ( C , D , ii) The fibronectin and laminin images were segmented to create 3D surface objects to characterize the volume and spacing. ( C , iii) The fibronectin images were further processed to create 3D filaments objects used to measure fiber diameter, length and orientation. ( F ) Hearts were also whole mounted in a 1:2 solution of benzyl alcohol to benzyl benzoate (BABB), a refractive index matching solution to clear the tissue. ( G ) 3D images of the whole heart, showing detailed trabeculae, were obtained using a macrozoom confocal microscope. Scale is in micrometers.

Article Snippet: To do this we created “filaments” (3D objects created by Imaris using a threshold as well as skeletonization to segment fibrous structures) and extracted information on diameter, length, and orientation.

Techniques: Staining, Confocal Microscopy, Refractive Index, Microscopy

Journal: Cells

Article Title: Extracellular Matrix Structure and Composition in the Early Four-Chambered Embryonic Heart

doi: 10.3390/cells9020285

Figure Lengend Snippet: Imaging of the myocardium of the left ventricle at days 5, 7, and 9 of development. ( A – C ) Actin staining of the aligned cardiomyocyte cytoskeleton at days 5, 7, and 9 (red = actin, blue = nuclei). ( D – F ) Images of the fibronectin matrix (green) shows ( D ) very thin fibronectin fibers at day 5, ( E ) emergence of some larger bundles (marked by white stars) around preliminary capillary lumens at day 7, and ( F ) an interconnected network of fibronectin-positive tubes at day 9. ( G – I ) Magnified images of the areas marked by dashed white boxes in ( D – F ) show how the fibronectin matrix is organized around the cardiomyocytes. ( G ) At day 5, small fibronectin fibers are regularly located between cardiomyocytes, following the main direction of alignment. ( H , I ) At day 7 and day 9, the large bungles of fibronectin around the developing capillaries are also following the main cardiomyocyte orientation. ( J – L ) 3D rendering of the fibronectin shows: ( J ) at day 5, very small filaments with a main direction of alignment; ( K ) at day 7, the formation of much longer fibronectin structures with what appears to be poorly defined lumens; ( L ) at day 9, fibronectin highlighting what appears to be well-aligned capillaries. Scale bars are 20 µm ( A – F ) and 10 µm ( G – I ).

Article Snippet: To do this we created “filaments” (3D objects created by Imaris using a threshold as well as skeletonization to segment fibrous structures) and extracted information on diameter, length, and orientation.

Techniques: Imaging, Staining

Journal: Cells

Article Title: Extracellular Matrix Structure and Composition in the Early Four-Chambered Embryonic Heart

doi: 10.3390/cells9020285

Figure Lengend Snippet: Quantitative analysis of fibronectin and laminin in the myocardium at 5 days. ( A ) ECM structures segmented using Imaris into “surfaces” were sorted into small, medium, or large groups depending on their volume. The ECM structures in each group are highlighted in yellow in 3D representations of fibronectin (green) and laminin (red). Scale is in micrometers. ( B ) Volume distribution of fibronectin and laminin, the dashed bars indicate the relative volume of each component (*, p < 0.01 determined by t-test). ( C ) The fibronectin image (green) was processed to find the distance of all points in the image from the fibronectin “surface,” where the value of each voxel is equal to the closest distance of the voxel to a fibronectin “surface.” The local maxima is a 2D image with all points equidistant from fibronectin “surfaces” highlighted in purple. Scale bars are 4 µm. ( D ) Histogram of distance to fibronectin showing mean ± standard deviation for each bin across all samples ( n = 11). ( E )The laminin image (red) was processed to find the distance of all points in the image from the laminin “surface,” where the value of each voxel is equal to the closest distance of the voxel to a laminin “surface.” The local maxima is a 2D image with all points equidistant from laminin “surfaces” highlighted in light blue. Scale bars are 4 µm. ( F ) Histograms of distance to laminin showing mean ± standard deviation for each bin across all samples ( n = 6). ( G – I ) Histograms of fibronectin fiber diameter, length, and orientation angle. fibronectin fibers were aligned in the main direction of myofiber orientation (green line) at 90°.

Article Snippet: To do this we created “filaments” (3D objects created by Imaris using a threshold as well as skeletonization to segment fibrous structures) and extracted information on diameter, length, and orientation.

Techniques: Standard Deviation