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In this Perspective the authors highlight and discuss the artifacts that can arise when using immunolabeling to examine protein localization in cell culture. They call for using both alternative fixation and permeabilization protocols and live-cell imaging of fluorescent protein fusions to reliably study subcellular protein localization.
In this Review, the authors take the reader through the steps needed to analyze bisulfite-treated DNA, pointing out different considerations for data in base or color space, to ensure high-quality methylome analysis.
Lipidic sponge phase crystallization yields membrane protein microcrystals that can be injected into an X-ray free electron laser beam, yielding diffraction patterns that can be processed to recover the crystal structure.
Molecular processes in cells are not spatially homogenous. Reported here is an approach, implemented in the Simmune toolset, for modeling cellular processes within their dynamic spatial context.
Expression of a protein in Sf9 insect cells at high concentration triggers formation of in vivo crystals that can be analyzed by serial femtosecond X-ray crystallography.
An imaging chamber implanted over the mouse spinal cord enables long-term longitudinal two-photon microscopy of cellular dynamics in normal or pathological conditions.
The authors report Alexa Fluor 633 hydrazide to be artery-specific and use it to measure arteriole dilation dynamics in vivo in response to visual stimuli in mouse, rat and cat neocortex. They find that sensory stimulus–evoked arteriole dilation reduces the fluorescence recorded from underlying neurons.
Automated tissue sectioning and two-photon imaging of fluorescently labeled and fixed mouse brains allows high-resolution tomographic imaging of the entire brain. The authors demonstrate performance using multiple GFP mouse lines, dye-based retrograde tracing and viral anterograde tracing.
A device for generating precise spatial and temporal patterns of airborne odorants is reported. In combination with machine vision tracking software, the authors use the device to monitor navigation of freely moving Drosophila melanogaster larvae.
The use of dual-objective detection with astigmatism-based three-dimensional stochastic optical reconstruction microscopy (STORM) imaging improves resolution more than twofold and removes noise in resulting super-resolution images. This allowed detailed fluorescence imaging of distinctive features of the three-dimensional actin cytoskeleton ultrastructure with single-filament resolution in cells.
A simple, general procedure for transferring protein complexes directly from native gels to electron microscopy grids for structural analysis is reported.
An acousto-optic two-photon microscope with continuous three-dimensional trajectory and random-access scanning modes can scan near-cubic-millimeter volumes of tissue at sub-millisecond temporal resolution in vivo. The system can be used to image both sub-cellular as well as network-scale neuronal activity.
