Research Article: Subcellular Microanatomy by 3D Deconvolution Brightfield Microscopy: Method and Analysis Using Human Chromatin in the Interphase Nucleus

Date Published: January 24, 2012

Publisher: Hindawi Publishing Corporation

Author(s): Paul Joseph Tadrous.


Anatomy has advanced using 3-dimensional (3D) studies at macroscopic (e.g., dissection, injection moulding of vessels, radiology) and microscopic (e.g., serial section reconstruction with light and electron microscopy) levels. This paper presents the first results in human cells of a new method of subcellular 3D brightfield microscopy. Unlike traditional 3D deconvolution and confocal techniques, this method is suitable for general application to brightfield microscopy. Unlike brightfield serial sectioning it has subcellular resolution. Results are presented of the 3D structure of chromatin in the interphase nucleus of two human cell types, hepatocyte and plasma cell. I show how the freedom to examine these structures in 3D allows greater morphological discrimination between and within cell types and the 3D structural basis for the classical “clock-face” motif of the plasma cell nucleus is revealed. Potential for further applications discussed.

Partial Text

Anatomical research requires methods for seeing structure in 3D such as the traditional macroscopic methods of dissection or making vascular casts by injection moulding [1, 2]. At the microscopic level, while equivalent techniques at the microscopic level do exist [3], it is more common for 3D microscopy to employ serial section reconstruction at light and electron microscope levels [4, 5] or some form of optical sectioning microscopy such as confocal or deconvolution fluorescence microscopy [6, 7].

Figure 1 shows a 3D rendering of an Z-stack of a hepatocyte nucleus with surrounding cytoplasm and it has a quarter of the volume “cut-away” for clarity and is shown at two angles. On the left is the original Z-stack prior to deconvolution and on the right is the deconvolved volume showing the greater nuclear chromatin detail in 3D following deconvolution.

This paper has demonstrated the first results in human cells of a novel method of 3D brightfield light microscopy in the detailed imaging of nuclear chromatin with the formation of high resolution and fully interactive 3D models of the interphase nucleus. The method is applicable to routinely stained paraffin-embedded tissue sections. The models generated from 6 hepatocyte and 6 plasma cell nuclei were studied quantitatively and qualitatively and it was shown that the 3D information can give greater morphological discriminatory information than 2D sections/projections. Furthermore, study of the plasma cell chromatin in high resolution 3D resulted in a plausible microanatomical explanation of the characteristic clock-face nucleus that typifies plasma cells in 2D preparations.