A simple technique of image analysis for specific nuclear immunolocalization of proteins (original) (raw)
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Journal of Neuroscience Methods, 1999
We describe here an automated system that accurately maps tissue sections stained by immunocytochemistry for an inducible nuclear protein. The sections are scanned with a computer-controlled microscope setup hooked to a CCD camera. Raw images captured at high resolution are filtered using highly selective criteria for the recognition of labeled cell nuclei. The total population of recognized labeled nuclei is then divided into separate bins, according to their labeling intensities. Finally, information about both the position and labeling intensity of labeled nuclei is represented in average density maps. The system was optimized for the quantitative mapping of neuronal cells expressing the inducible gene ZENK in the brain of songbirds, in response to stimulation with song, but should be of general applicability for the mapping of inducible nuclear proteins.
BioTechniques, 2004
Many nuclear proteins, including the nuclear receptor co-repressor (NCoR) protein are localized to specific regions of the cell nucleus, and this subnuclear positioning is preserved when NCoR is expressed in cells as a fusion to a fluorescent protein (FP). To determine how specific factors may influence the subnuclear organization of NCoR requires an unbiased approach to the selection of cells for image analysis. Here, we use the co-expression of the monomeric red FP (mRFP) to select cells that also express NCoR labeled with yellow FP (YFP). The transfected cells are selected for imaging based on the diffuse cellular mRFP signal without prior knowledge of the subnuclear organization of the co-expressed YFP-NCoR. The images acquired of the expressed FPs are then analyzed using an automated image analysis protocol that identifies regions of interest (ROIs) using a set of empirically determined rules. The relative expression levels of both fluorescent proteins are estimated, and YFP-NC...
Cytometry, 2002
Background: Visualization of more than one antigen by multicolor immunostaining is often desirable or even necessary to explore spatial and temporal relationships of functional significance. Previously presented staining protocols have been limited to the visualization of three or four antigens. Methods: Immunofluorescence staining was performed both on slices of formalin-fixed tissue and on cells in culture. Images of the stained material were recorded using digital imaging fluorescence microscopy. The primary and secondary antibodies, as well as the fluorophores, were thereafter removed using a combination of denaturation and elution techniques. After removal of the fluorescence stain, a new immunofluorescence staining was performed, visualizing a new set of antigens. The procedure was repeated up to three times. A method for image registration combined with segmentation, extraction of data, and cell classification was developed for efficient and objective analysis of the image data.
Journal of Immunological Methods, 1981
Some examples are given of immunofluorescence with tissue sections and microtubular cytoskeletons of cultured cells where the fluorescent dye propidium iodide (PI) has been used as marker of nuclei. The emission wave length of PI is longer than that of fluorescein, making it possible to use several different and commonly available filter combinations. The use of nuclei as positional indicators is often a more suitable method than phase microscopy combined with immunofluorescence because of low background illumination against which morphology is viewed, circumventing the need for often expensive phase optics.
Cytometry, 2001
The recently developed technique of highresolution cytometry (HRCM) enables automated acquisition and analysis of fluorescent in situ hybridization (FISH)-stained cell nuclei using conventional wide-field fluorescence microscopy. The method has now been extended to confocal imaging and offers the opportunity to combine the advantages of confocal and wide-field modes. Methods: We have automated image acquisition and analysis from a standard inverted fluorescence microscope equipped with a confocal module with Nipkow disk and a cooled digital CCD camera. The system is fully controlled by a high-performance computer that performs both acquisition and related on-line image analysis. The system can be used either for an automatic two (2D) and three-dimensional (3D) analysis of FISH-stained interphase nuclei or for a semiautomatic 3D analysis of FISHstained cells in tissues. The user can select which fluorochromes are acquired using wide-field mode and which using confocal mode. The wide-field and confocal images are overlaid automatically in computer memory. The developed software compensates automatically for both chromatic color shifts and spatial shifts caused by switching to a different imaging mode.
The Histochemical …, 1993
A widely applicable method for the accurate quantification or semiquantification of macromolecules at the level of individual cells is described and validated; this is a method which may considerably facilitate the study of many biological processes. This method relies on measuring fluorescent emission in immunocytochemically labelled cells with a confocal microscope. Emission is related quantitatively to the level of the fluorophore by the combination of an analysis of the polarization of the fluorescent emission and fluorophore ratioing methods. The method .was applied to the study of the expression of the suppressed cyclic AMP-induced POU protein (SCIP) transcription factor in glial cells of the central nervous system. In particular, the method allowed the study of transcription factor expression in defined cells present in heterogeneous cultures and in cell types which cannot be isolated in sufficient numbers for biochemical analysis using conventional techniques.
Chromosome Research, 2008
The vast majority of microscopic data in biology of the cell nucleus is currently collected using fluorescence microscopy, and most of these data are subsequently subjected to quantitative analysis. The analysis process unites a number of steps, from image acquisition to statistics, and at each of these steps decisions must be made that may crucially affect the conclusions of the whole study. This often presents a really serious problem because the researcher is typically a biologist, while the decisions to be taken require expertise in the fields of physics, computer image analysis, and statistics. The researcher has to choose between multiple options for data collection, numerous programs for preprocessing and processing of images, and a number of statistical approaches. Written for biologists, this article discusses some of the typical problems and errors that should be avoided. The article was prepared by a team uniting expertise in biology, microscopy, image analysis, and statistics. It considers the options a researcher has at the stages of data acquisition (choice of the microscope and acquisition settings), preprocessing (filtering, intensity normalization, deconvolution), image processing (radial distribution, clustering, co-localization, shape and orientation of objects), and statistical analysis.
Three-dimensional immunogold labeling of nuclear matrix proteins in permeabilized cells
Cell Biology International Reports, 1992
A preembedment labeling procedure is described for the three-dimensional (3D) labeling of nuclear matrix proteins in penneabilized ceils. The procedure is based on the use of ultra-small (1 nm) gold particles as a marker system. This marker penetrates the nucleus more efficiently than the conventionally used 5-10 nm colloidal gold probes. Dehydration is performed by freeze-substitution to preserve the ultrastructure of the cell as optimally as possible. During freeze-substitution the samples are stained by uranyl ions to stain the cellular material throughout the resin section. The 3D gold-labeled and uranyl-stained specimen is embedded in Epon resin and semi-thin (0.2-0.5 1am) sections are made for stereo electron microscopy. The applicability of this method is illustrated by the localization of nuclear matrixassociated nuclear bodies in permeabilized interphase and mitotic HeLa cells.
Diagnostic Pathology, 2013
This paper describes the multistage morphological segmentation method (MSMA) for microscopic cell images. The pro− posed method enables us to study the cell behaviour by using a sequence of two types of microscopic images: bright field images and/or fluorescent images. The proposed method is based on two types of information: the cell texture coming from the bright field images and intensity of light emission, done by fluorescent markers. The method is dedicated to the image sequences segmentation and it is based on mathematical morphology methods supported by other image processing tech− niques. The method allows for detecting cells in image independently from a degree of their flattening and from presenting structures which produce the texture. It makes use of some synergic information from the fluorescent light emission image as the support information. The MSMA method has been applied to images acquired during the experiments on neural stem cells as well as to artificial images. In order to validate the method, two types of errors have been considered: the error of cell area detection and the error of cell position using artificial images as the "gold standard".