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

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. 1. Introduction 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]. In order to overcome some of the limitations of traditional deconvolution and confocal microscopy (which are generally restricted to fluorescent preparations with a few special case exceptions, especially if subcellular detail is required), recently a new method of 3D deconvolution microscopy has been published [8]. This method was designed specifically for brightfield microscopy to make visible routinely stained histological preparations with subcellular 3D resolution using an ordinary light microscope and results on yeast cells were presented [8]. In this paper I present, for the first time, results using this new method applied to human cells, specifically hepatocytes and plasma cells. I show how the method reveals the interphase nuclear chromatin meshwork in 3D. The results are analysed quantitatively with texture analysis and qualitatively to demonstrate the utility of this method in the study of cell and tissue structure. 2. Materials and Methods 2.1. Tissue Preparation The cells analysed in this study were imaged from a formalin-fixed paraffin-embedded 10-micron-thick section of human liver