Mapping of cellular compartments based on ultrastructural immunogold labeling.
Ultrastructural identification of subcellular morphologically inconspicuous compartments is based on detection of specific molecules or by a presence of specific functions. Such compartments are detected using antibodies with attached label, usually gold particles. However, the gold particles have a point pattern, while a compartment is a coherent area. In addition, some background labeling is always present that complicates identification of the labeled compartments. The aim of this study was therefore to develop a stereological method that would enable us to define cellular compartments based on delineating the borders of gold particle clusters, and to test the practical use of the method using biological experimental data. New computer program plug-ins were developed to facilitate the practical use of the stereological method. The kernel estimation method was successfully tested by detection of ribosomal rRNA over morphologically recognizable nucleoli. In a next step, we successfully detected individual chromosomal domains-nuclear compartments that cannot be distinguished in cell nuclei morphologically. The results show that the new stereological/image analysis method is well able to discriminate cellular compartments based on density of immunogold particles. The plug-ins were made available to scientific community at http://nucleus.biomed.cas.cz/gold.
Schöfer
Christian
C
Institute for Histology and Embryology, Medical University Vienna, Vienna, Austria.
Janácek
Jirí
J
Weipoltshammer
Klara
K
Pourani
Jamile
J
Hozák
Pavel
P
D002451:Cell Compartmentation; D002877:Chromosomes, Human / Q000737:chemistry; D006046:Gold / Q000737:chemistry; D006367:HeLa Cells; D006801:Humans; D021621:Imaging, Three-Dimensional; D007150:Immunohistochemistry; D017403:In Situ Hybridization; D016253:Microscopy, Immunoelectron / Q000379:methods*; D012335:RNA, Ribosomal / Q000032:analysis / Q000737:chemistry; D012984:Software
D012335:RNA, Ribosomal; D006046:Gold