Optical properties of biological tissues: a review. Assessing phototoxicity in live fluorescence imaging. Optimizing live-cell fluorescence imaging conditions to minimize phototoxicity. Kiepas, A., Voorand, E., Mubaid, F., Siegel, P. Advanced light microscopy core facilities: balancing service, science and career. Synthesis of Janelia Fluor HaloTag and SNAP-Tag ligands and their use in cellular imaging experiments. A general strategy to develop cell permeable and fluorogenic probes for multicolour nanoscopy.

Labeling strategies matter for super-resolution microscopy: a comparison between HaloTags and SNAP-tags. Two-colour live-cell nanoscale imaging of intracellular targets.

Quantitative assessment of fluorescent proteins. The growing and glowing toolbox of fluorescent and photoactive proteins. Engineering and characterizing monomeric fluorescent proteins for live-cell imaging applications. FPbase: a community-editable fluorescent protein database. Photoconversion of DAPI following UV or violet excitation can cause DAPI to fluoresce with blue or cyan excitation. Mechanisms and advancement of antifading agents for fluorescence microscopy and single-molecule spectroscopy. Quality assurance testing for modern optical imaging systems. Systematic validation of antibody binding and protein subcellular localization using siRNA and confocal microscopy. A review of reagents for fluorescence microscopy of cellular compartments and structures, Part II: reagents for non-vesicular organelles. A review of reagents for fluorescence microscopy of cellular compartments and structures, part I: BacMam labeling and reagents for vesicular structures. A simple, versatile method for GFP-based super-resolution microscopy via nanobodies. Ries, J., Kaplan, C., Platonova, E., Eghlidi, H. Advances in chemical labeling of proteins in living cells. Implications for immunofluorescence microscopy. Redistribution and differential extraction of soluble proteins in permeabilized cultured cells. Glyoxal as an alternative fixative to formaldehyde in immunostaining and super-resolution microscopy. Protection of tissue physicochemical properties using polyfunctional crosslinkers. Immunocytochemistry: A Practical Guide for Biomedical Research (Springer, 2010). Distinct desmocollin isoforms occur in the same desmosomes and show reciprocally graded distributions in bovine nasal epidermis. Recent advances in high-pressure freezing: equipment- and specimen-loading methods. in Protein Localization by Fluorescence Microscopy: A Practical Approach (ed. Aberrations in confocal fluorescence microscopy induced by mismatches in refractive index. Seeing is believing? A beginners’ guide to practical pitfalls in image acquisition. The 39 steps: a cautionary tale of quantitative 3-D fluorescence microscopy. A visual summary of this tutorial is available as a poster ( ). Finally, guidelines for analyzing and presenting confocal images in a way that maintains the quantitative nature of the data are presented, and statistical analysis is discussed. Common pitfalls such as photobleaching and cross-talk are addressed, as well as several troubling instrumentation problems that may prevent the acquisition of quantitative data. Suggestions are offered for planning unbiased and rigorous confocal microscope experiments. In this tutorial, the researcher is guided through all aspects of acquiring quantitative confocal microscopy images, including optimizing sample preparation for fixed and live cells, choosing the most suitable microscope for a given application and configuring the microscope parameters. However, generating meaningful data using confocal microscopy requires careful planning and a thorough understanding of the technique. The confocal microscope’s ability to block out-of-focus light and thereby perform optical sectioning through a specimen allows the researcher to quantify fluorescence with very high spatial precision. When used appropriately, a confocal fluorescence microscope is an excellent tool for making quantitative measurements in cells and tissues.

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