Combined light and fluorescent microscopical imaging of nucleolar organizer regions and cellular rRNA as detected by fluorescent in situ hybridization

Abstract

A method for combined light and fluorescent microscopic imaging of nucleolar organizer regions and cellular rRNA is described. Nucleolar organizer regions were detected by silver staining (Ag-NOR), and rRNA was detected by fluorescent in situ hybridization (FISH). MG-63 human fibrosarcoma cells were silver stained prior to in situ hybridization. To quantitate Ag-NOR within individual cells, brightfield images were digitized, and the total Ag-NOR arednucleus was determined. Fluorescent images were digitized, and the total cellular fluorescence was calculated after correction for nonuniformity of illumination. By using this method, it was shown that the Ag-NOR procedure did not signiticantly affect the fluorescence intensity related to FISH. Furthermore, the hybridization procedure did not interfere with quantitation of Ag-NOR. With this method, both Ag-NOR and rRNA product can be quantitated within the same cell. Because the relationship of rRNA content to cell proliferation is well established, correlation to quantitative Ag-NOR parameters within individual cells will contribute to the better definition of the relationship of quantitative Ag-NOR indices with cellular proliferation. 0 1995 Wiley-Liss, Inc.

Pipeline for illumination correction of images for high‐throughput microscopy

“…Thus, in practice, images acquired from standard or automated microscopes, even with white referencing, are generally adequate for visual inspection but unsatisfactory for quantitative image analysis, because the intensity of an object is rendered dependent on its position within the field of view. For example, in careful studies, corrections using a white reference image of a uniformly fluorescent dye reduced the variation in mean intensity of a cell's signal from ∼20% to ∼5% (Pajor & Honeyman, ) and from ∼30% to ∼5% (Model & Burkhardt, ). In practical use, when conditions are not so carefully controlled, differences can be more substantial; in our experience, illumination can routinely vary 10–30% across a single image when using standard microscope hardware, even when white‐referencing has been applied (see below).…”

Philadelphia chromosome and/or bcr‐abl mRNA‐positive primary thrombocytosis: morphometric evidence for the transition from essential thrombocythaemia to chronic myeloid leukaemia type of myeloproliferation

“…Furthermore, we presumed that a disorder like essential thrombocythaemia, characterized by major thrombocytosis, also differs from typical chronic myeloid leukaemia in the activity of megakaryocytes represented by the total NOR area ⁄ nucleus. 12,24 Indeed, statistical analysis yielded significant differences between essential thrombocythaemia and chronic myeloid leukaemia in all three parameters, i.e. the bone marrow is less cellular and the megakaryocytes are larger and more active in essential thrombocythaemia.…”

“…Two of them, bone marrow cellularity and the size of megakaryocytes in essential thrombocythaemia versus chronic myeloid leukaemia, are well known for being different even at the level of morphological assessment. Furthermore, we presumed that a disorder like essential thrombocythaemia, characterized by major thrombocytosis, also differs from typical chronic myeloid leukaemia in the activity of megakaryocytes represented by the total NOR area/nucleus 12,24 . Indeed, statistical analysis yielded significant differences between essential thrombocythaemia and chronic myeloid leukaemia in all three parameters, i.e.…”

“…This value was obtained from the serial sections silver stained for NOR. The megakaryocytes were identified by morphology, and the total NOR area per megakaryocyte nucleus was obtained as previously described 12 …”