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At least 415 records · Page 23Linked to original sources
PubMed2004

Phase-retrieved pupil functions in wide-field fluorescence microscopy.

Pupil functions are compact and modifiable descriptions of the three-dimensional (3D) imaging properties of wide-field optical systems. The pupil function of a microscope can be computationally estimated from the measured point spread function (PSF) using phase retrieval algorithms. The compaction of a 3D PSF into a 2D pupil function suppresses artefacts and measurement noise without resorting to rotational averaging. We show here that such 'phase-retrieved' pupil functions can reproduce features in the optical path, both near the sample and in the microscope. Unlike the PSF, the pupil function can be easily modified to include known aberrations, such as those induced by index-mismatched mounting media, simply by multiplying the pupil function by a calculated aberration function. PSFs calculated from such a modified pupil function closely match the corresponding measured PSFs collected under the aberrated imaging conditions. When used for image deconvolution of simulated objects, these phase-retrieved, calculated PSFs perform similarly to directly measured PSFs.

Algorithms↗
PubMed2005

Noise suppression of point spread functions and its influence on deconvolution of three-dimensional fluorescence microscopy image sets.

The point spread function (PSF) is of central importance in the image restoration of three-dimensional image sets acquired by an epifluorescent microscope. Even though it is well known that an experimental PSF is typically more accurate than a theoretical one, the noise content of the experimental PSF is often an obstacle to its use in deconvolution algorithms. In this paper we apply a recently introduced noise suppression method to achieve an effective noise reduction in experimental PSFs. We show with both simulated and experimental three-dimensional image sets that a PSF that is smoothed with this method leads to a significant improvement in the performance of deconvolution algorithms, such as the regularized least-squares algorithm and the accelerated Richardson-Lucy algorithm.

Algorithms↗
PubMed1991

Automatic classification of cells in cell cycle phases based on Ki-67 antigen quantification by fluorescence microscopy.

Ki-67 antigen is thought to be a marker of cell proliferation, as it can be detected in cycling cells, i.e. cells in G1, S, G2 and M phases, but not in resting cells. The immunocytochemical staining pattern obtained by the Ki-67 monoclonal antibody varies, depending on the cell cycle phases. Analysis of double staining of Ki-67 antigen and DNA in the MCF-7 cell line by videomicrofluorometry allows the description of both the level and the pattern of Ki-67 staining in the form of a set of parameters defining each cell. These parameters were measured in MCF-7 cell populations characterized according to their position in the cell cycle. They were submitted to a statistical analysis (principal component and discriminant analysis) which allowed the determination of the optimal parameters to characterize a given cellular group and permitted the use of these parameters for an automatic classification of cells in the different cell cycle phases. In G1, S, G2, prophase + metaphase and anaphase + telophase cells, these parameters allowed a classification of cells with a good-classification rate of 94.37%. A comparison of this method with methods based on the DNA histogram and bromodeoxyuridine uptake was performed. The classification coefficients stemming from the discriminant analysis were introduced into a program to obtain, automatically, the Ki-67 labelling index and the percentages of cells in each phase. This method, which allows a quick evaluation of the proliferation and the phase indices, may be more widely applicable.

Breast Neoplasms↗
PubMed2004

Fluorescence microscopy of colour-tagged nanoparticles that are undergoing thermal motion.

To bypass limitations of conventional biochemical analysis, single-particle biochemical analysis is used. To improve single-particle biochemical analysis, procedures are needed to keep nanometre-sized particles in focus while the particles are undergoing thermal motion. A simple, inexpensive procedure is developed here for keeping particles in focus during the continuous observing/discriminating/recording of two different particles, both of which are undergoing thermal motion. This procedure concentrates the particles in a plane of solution that is in focus when the cover glass surface is in focus. An essential component of the procedure is the addition of molten, low-melt agarose to the specimen. Motionless binding to glass is inhibited by inclusion of anti-stick additives in the specimen. Both carrier protein (gelatin) and non-ionic detergent (Triton X-100) are anti-stick additives successfully used here. Intact bacteriophages T3 and T7 are used as model particles, in anticipation of the use of the procedures developed here for the analysis of the assembly of bacteriophages. Observing/discriminating/recording of colour-tagged bacteriophages T3 and T7 is achieved at video frame rate with image splitting to discriminate colours.

Acetic Acid↗
PubMed1991

Anti-granulocyte antibodies (C-ANCA, P-ANCA, GS-ANA) studied by confocal scanning laser fluorescence microscopy, ELISA, and chemiluminescence techniques.

Fifty-nine patient sera with antibodies against human polymorphonuclear neutrophil granulocyte (PMN) antigens, as determined primarily by indirect immunofluorescence microscopy (IIF) screening, were further analysed by enzyme-linked immunosorbent assays (ELISA). The antibodies were primarily characterized by their immunomorphological staining patterns on ethanol-fixed PMN as judged by conventional IIF microscopy, i.e. anti-neutrophil cytoplasmic antibodies (ANCA) giving a pancytoplasmic granular staining pattern (C-ANCA) or a diffuse perinuclear cytoplasmic pattern (P-ANCA), or granulocyte-specific anti-nuclear antibodies (GS-ANA) producing a homogeneous or peripheral nuclear staining pattern. The three distinct patterns were confirmed by confocal scanning laser IIF microscopy. As antigen substrates in the ELISA tests we used an extract from azurophil PMN granules, myeloperoxidase (MPO), and lactoferrin. As expected, most (but not all) of the C-ANCA positive sera turned out positive in the alpha-ELISA assay. Both P-ANCA and GS-ANA positive sera had high frequencies of antibodies against MPO. Occasional P-ANCA positive sera contained antilactoferrin antibodies. Although P-ANCA and GS-ANA in general probably represent the same type of auto-antibodies, we regard it appropriate to make a distinction between the two patterns, until the existence of 'true' granulocyte-specific ANAs has been ruled out. All sera were analysed for their ability to activate PMN in vitro as judged by the generation of a chemiluminescence (CL) response. Sera containing C-ANCA, as well as sera containing P-ANCA or GS-ANA, showed high frequencies of positive CL tests using 'resting' isolated PMN. The reactions were diminished, but not always abolished, by heat-treatment of the sera.

Autoantibodies↗