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.

A guided tour into subcellular colocalization analysis in light microscopy

For centuries, cell biology has been based on light microscopy and at the same time been limited by its optical resolution. However, several new technologies have been developed recently that bypass this limit. These new super-resolution technologies are either based on tailored illumination, nonlinear fluorophore responses, or the precise localization of single molecules. Overall, these new approaches have created unprecedented new possibilities to investigate the structure and function of cells.

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A guide to super-resolution fluorescence microscopy

Since the earliest examination of cellular structures, biologists have been fascinated by observing cells using light microscopy. The advent of fluorescent labeling technologies plus the plethora of sophisticated light microscope techniques now available make studying dynamic processes in living cells almost commonplace. For anyone new to this area, however, it can be daunting to decide which techniques or equipment to try. Here, we aim to give a brief overview of the main approaches to live cell imaging, with some mention of their pros and cons.

https://science.sciencemag.org/content/sci/300/5616/82.full.pdf

Light Microscopy Techniques for Live Cell Imaging

Aurora B Regulates MCAK at the Mitotic Centromere

The light microscope has long been used to document the localization of fluorescent molecules in cell biology research. With advances in digital cameras and the discovery and development of genetically encoded fluorophores, there has been a huge increase in the use of fluorescence microscopy to quantify spatial and temporal measurements of fluorescent molecules in biological specimens. Whether simply comparing the relative intensities of two fluorescent specimens, or using advanced techniques like Forster resonance energy transfer (FRET) or fluorescence recovery after photobleaching (FRAP), quantitation of fluorescence requires a thorough understanding of the limitations of and proper use of the different components of the imaging system. Here, I focus on the parameters of digital image acquisition that affect the accuracy and precision of quantitative fluorescence microscopy measurements.

/pdf/accuracy-and-precision-in-quantitative-fluorescence-2g1hq10tvn.pdf

Accuracy and precision in quantitative fluorescence microscopy

The fluorescence microscope is routinel y used to study cellular structure in many bi o medical research laboratories and is i n creasingly used as a quantitative assay sy s tem for cellular dynamics. One of the majo r causes of image degradation in the fluore s cence microscope is blurring. Deconvol u tion algorithms use a model of the micr o scope imaging process to either subtract o r reassign out-of-focus blur. A variety of a l gorithms are now commercially available , each with its own characteristic advantage s and disadvantages. In this article, we r e view the imaging process in the fluore s cence microscope and then discuss how th e various deconvolution methods work. Fina l ly, we provide a summary of practical tip s for using deconvolution and discuss ima g ing artifacts and how to minimize them .

/pdf/a-workingperson-s-guide-to-deconvolution-in-light-microscopy-4alg3nsnf8.pdf

Lutz H. Schaefer

Papers

A Workingperson’s Guide to Deconvolution in Light Microscopy