Local Resolution Analysis
Local Resolution Analysis - Related Videos
12:28
Correlative Super-resolution and Electron Microscopy to Resolve Protein Localization in Zebrafish Retina
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Cited by 11 •
2017
This protocol describes the necessary steps to obtain subcellular protein localization results on zebrafish retina by correlating super-resolution light microscopy and scanning electron microscopy images.
10:36
Ultrasound Localization Microscopy for Super-Resolution Mapping of the Rodent Brain Microvasculature
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2025
Ultrasound Localization Microscopy (ULM) is a recently developed technique that allows for an unprecedented level of resolution in imaging the microvasculature in vivo. In this work, we describe in detail how to obtain super-resolved images of the brain microvasculature in rodents using a standardized functional ultrasound imaging platform designed for preclinical research.
11:57
Three-dimensional Super Resolution Microscopy of F-actin Filaments by Interferometric PhotoActivated Localization Microscopy (iPALM)
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Cited by 10 •
2016
We present a protocol for the application of interferometric PhotoActivated Localization Microscopy (iPALM), a 3-dimensional single-molecule localization super resolution microscopy method, to the imaging of the actin cytoskeleton in adherent mammalian cells. This approach allows light-based visualization of nanoscale structural features that would otherwise remain unresolved by conventional diffraction-limited optical microscopy.
13:28
High-resolution Single Particle Analysis from Electron Cryo-microscopy Images Using SPHIRE
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Cited by 218 •
2017
This paper presents a protocol for processing cryo-EM images using the software suite SPHIRE. The present protocol can be applied for nearly all single particle EM projects that target near-atomic resolution.
11:24
Detection of Axonally Localized mRNAs in Brain Sections Using High-Resolution In Situ Hybridization
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Cited by 8 •
2015
RNA in situ hybridization (ISH) enables the visualization of RNAs in cells and tissues. Here we show how combination of RNAscope ISH with immunohistochemistry or histological dyes can be successfully used to detect mRNAs localized to axons in sections of mouse and human brains.
FAQs
Fourier shell correlation (FSC) provides a quantitative comparison between independently generated half-maps. For each localized volume, the analysis examines how consistently the two reconstructions contain signal across spatial frequency shells. Stronger agreement supports finer detail in that region, whereas weaker agreement indicates poorer local signal quality. This comparison makes spatial differences visible within the same molecular reconstruction.
Resolution varies because structural regions do not always contribute equally well-ordered or consistent signal. Flexible domains, mobile loops, compositional heterogeneity, and poorly ordered regions can reduce the clarity of particular localized volumes. Other parts of the molecule may remain better resolved. Recognizing this variation helps distinguish reliable structural features from regions requiring more cautious interpretation.
A single overall value can conceal substantial differences between regions of the same map. Local Resolution Analysis identifies where structural detail is clearer or less certain, allowing interpretation to reflect spatial variation in signal quality. This distinction matters when evaluating individual domains, loops, or other features whose visibility may differ from the reconstruction as a whole.
The workflow begins by dividing a three-dimensional reconstruction into localized volumes. Independently generated half-maps are then compared within those volumes, commonly using Fourier shell correlation to estimate the resolution of each region. The resulting values are assembled into a resolution map, which displays spatial variation and can be examined alongside the molecular reconstruction.
A resolution map helps researchers judge which portions of a reconstruction provide sufficiently clear structural information for confident model building. Better-resolved regions may support more detailed interpretation, while poorly resolved areas signal the need for caution. By linking modeling decisions to local signal quality, the analysis helps prevent one global resolution estimate from being applied uniformly.
In biochemical structures determined by cryo-electron microscopy, local resolution patterns can highlight flexible domains, mobile loops, compositional heterogeneity, and poorly ordered regions. These patterns help relate structural clarity to molecular organization and behavior. They also indicate which conclusions about a molecule are most reliable, supporting more careful interpretation of domains and other localized features.