Three-dimensional direct measurement of cardiomyocyte volume, nuclearity, and ploidy in thick histological sections

Abstract

Quantitative assessment of myocardial development and disease requires accurate measurement of cardiomyocyte volume, nuclearity (nuclei per cell), and ploidy (genome copies per cell). Current methods require enzymatically isolating cells, which excludes the use of archived tissue, or serial sectioning. We describe a method of analysis that permits the direct simultaneous measurement of cardiomyocyte volume, nuclearity, and ploidy in thick histological sections. To demonstrate the utility of our technique, heart tissue was obtained from four species (rat, mouse, rabbit, sheep) at up to three life stages: prenatal, weaning and adulthood. Thick (40 μm) paraffin sections were stained with Wheat Germ Agglutinin-Alexa Fluor 488 to visualise cell membranes, and DAPI (4',6-diamidino-2-phenylindole) to visualise nuclei and measure ploidy. Previous methods have been restricted to thin sections (2-10 μm) and offer an incomplete picture of cardiomyocytes. Using confocal microscopy and three-dimensional image analysis software (Imaris Version 8.2, Bitplane AG, Switzerland), cardiomyocyte volume, nuclearity, and ploidy were measured. This method of staining and analysis of cardiomyocytes enables accurate morphometric measurements in thick histological sections, thus unlocking the potential of archived tissue. Our novel time-efficient method permits the entire cardiomyocyte to be visualised directly in 3D, eliminating the need for precise alignment of serial sections.

Figure 1

(A) Cardiomyocytes in long section, DAPI appears in blue, WGA-AF488 appears in green. (B) Cardiomyocytes in cross-section, DAPI appears in blue, WGA-AF488 appears in grey.

Figure 2

(A,B) Representative mean intensity chart. (A) represents the mean intensity through the z-stack for DAPI. (B) represents the mean intensity through the z-stack for WGA-AF488. The X axis represents the depth through the section (z-stack), the Y axis is the mean intensity per channel at each depth (frame of the z-stack).

Figure 3. Cardiomyocyte example. 3.5 μm pitch across 12 sections (42 μm coverage).

DAPI appears in yellow/red, WGA-AF488 appears in green. *denotes the example cell analysed in Fig. 4. Scale bar represents 25 μm.

Figure 4. Cardiomyocyte delineation example.

The selected cardiomyocyte is in green. In the top left hand corner is the Z-stack position, in the top right hand corner is the X axis projection. In the bottom left hand corner is the Y axis projection. Seed was laid down at the cross-hair point.

Figure 5. Alternative method for cardiomyocyte volume measurement.

In (panel A) WGA-AF488 staining appears in green. (Panel B) shows a brightness inversion of the left panel. In (panel C) the image was binarised and manual clean-up performed to produce dark cytoplasm and bright membranes. In (panel D) the composite image including both the WGA-AF488 (again in green) and DAPI is shown (in blue).

Figure 6. Z-stack through the left ventricle of a fetal sheep.

333 nm pitch across 4 μm of total coverage. DAPI appears in red, WGA-AF488 appears in green. Mitotic nucleus appears in the white circles.

Figure 7

(A,B) Identification of capillaries by WGA-AF488 enhancing staining. On the left (Panel A) DAPI appears in yellow/red, WGA-AF488 appears in green. On the right (Panel B) DAPI appears in blue, WGA-AF488 appears in green. Arrows identify capillaries.

Figure 8. Brightest point projection method to demonstrate the 3-dimensional appearance of capillaries in the heart, from the same z-stack as Fig. 7A.