Journal of Microscopy 

About: Journal of Microscopy is an academic journal published by Wiley-Blackwell. The journal publishes majorly in the area(s): Microscope & Microscopy. It has an ISSN identifier of 0022-2720. Over the lifetime, 6868 publications have been published receiving 222540 citations. The journal is also known as: Monthly microscopical journal & Transactions of the Royal Microscopical Society.

A guided tour into subcellular colocalization analysis in light microscopy

Abstract: Summary It is generally accepted that the functional compartmentalization of eukaryotic cells is reflected by the differential occurrence of proteins in their compartments. The location and physiological function of a protein are closely related; local information of a protein is thus crucial to understanding its role in biological processes. The visualization of proteins residing on intracellular structures by fluorescence microscopy has become a routine approach in cell biology and is increasingly used to assess their colocalization with well-characterized markers. However, imageanalysis methods for colocalization studies are a field of contention and enigma. We have therefore undertaken to review the most currently used colocalization analysis methods, introducing the basic optical concepts important for image acquisition and subsequent analysis. We provide a summary of practical tips for image acquisition and treatment that should precede proper colocalization analysis. Furthermore, we discuss the application and feasibility of colocalization tools for various biological colocalization situations and discuss their respective strengths and weaknesses. We have created a novel toolbox for subcellular colocalization analysis under Image J, named JACoP, that integrates current global statistic methods and a novel object-based approach.

The efficiency of systematic sampling in stereology and its prediction

Abstract: The superior efficiency of systematic sampling at all levels in stereological studies is emphasized and various commonly used ways of implementing it are briefly described. Summarizing recent theoretical and experimental studies a set of very simple estimators of efficiency are presented and illustrated with a variety of biological examples. In particular, a nomogram for predicting the necessary number of points when performing point counting is provided. The very efficient and simple unbiased estimator of the volume of an arbitrary object based on Cavalieri's principle is dealt with in some detail. The efficiency of the systematic fractionating of an object is also illustrated.

Surpassing the lateral resolution limit by a factor of two using structured illumination microscopy.

Abstract: Lateral resolution that exceeds the classical diffraction limit by a factor of two is achieved by using spatially structured illumination in a wide-field fluorescence microscope. The sample is illuminated with a series of excitation light patterns, which cause normally inaccessible high-resolution information to be encoded into the observed image. The recorded images are linearly processed to extract the new information and produce a reconstruction with twice the normal resolution. Unlike confocal microscopy, the resolution improvement is achieved with no need to discard any of the emission light. The method produces images of strikingly increased clarity compared to both conventional and confocal microscopes.

The unbiased estimation of number and sizes of arbitrary particles using the disector

Abstract: A three-dimensional counting rule and its integral test system, the disector, for obtaining unbiased estimates of the number of arbitrary particles in a specimen is presented. Used in combination with ordinary and recently developed stereological methods unbiased estimates of various mean particle sizes and the variance of particle volume are obtainable on sets of two parallel sections with a known separation. The same principle allows the unbiased estimation of the distribution of individual particle volumes in sets of serial sections.

Measurement of co-localization of objects in dual-colour confocal images

Abstract: A method to measure the degree of co-localization of objects in confocal dual-colour images has been developed. This image analysis produced two coefficients that represent the fraction of co-localizing objects in each component of a dual-channel image. The generation of test objects with a Gaussian intensity distribution, at well-defined positions in both components of dual-channel images, allowed an accurate investigation of the reliability of the procedure. To do that, the co-localization coefficients were determined before degrading the image with background, cross-talk and Poisson noise. These synthesized sources of image deterioration represent sources of deterioration that must be dealt with in practical confocal imaging, namely dark current, non-specific binding and cross-reactivity of fluorescent probes, optical cross-talk and photon noise. The degraded images were restored by filtering and cross-talk correction. The co-localization coefficients of the restored images were not significantly different from those of the original undegraded images. Finally, we tested the procedure on images of real biological specimens. The results of these tests correspond with data found in the literature. We conclude that the co-localization coefficients can provide relevant quantitative information about the positional relation between biological objects or processes.