Confocal Microscopic 3D Visualization of Membranous Organelles in Living Cultured Fibroblasts

Abstract

The 3D distribution of membranous organelles in living cultured cells was visualized by confocal laser scanning microscopy combined with lipophilic probe staining. When cultured 3Y1 fibroblasts were stained with a fluorescent dicarbocyanine, DiOC₆(3), mitochondria, endoplasmic reticulum and plasmalemma were visualized at high resolution under a confocal laser scanning microscope. Stereo pair images reconstructed from serial optical sections of such stained cells clearly showed the 3D distribution of mitochondria and endoplasmic reticulum throughout the cytoplasm. Microvilli were also seen to protrude from the cell surface. BODIPY-conjugated ceramide selectively accumulated in the Golgi apparatus to provide sharp 3D images of the organelle. For counter-staining of such stained living cells, we used dihydroethidium which selectively stained nuclei and chromosomes. We conclude that confocal 3D microscopy combined with lipophilic probe staining is a useful approach for better understanding not only of the 3D distribution but also of the dynamic features of membranous organelles in living cells.