3dgenome
  • Initial page
  • Cover
  • Preface
  • Figurelist
  • Chap0 Preparation
    • 0.1 Molecular biology
    • 0.2 Sequencing technologies
    • 0.3 RNA-seq Data Mapping & Gene Quantification
    • 0.4 RNA-seq Differential Analysis
  • Chap1 Why we care about 3D genome
    • 1.1 From 2D to 3D nuclear structure
    • 1.2 From static to dynamic
    • 1.3 From intra to inter chromosomes "talk"
    • 1.4 From aggregation to division - phase separation
  • Chap2 experiment tools for exploring genome interaction
    • 2.1 Image based
    • 2.2 Primary order
    • 2.3 Higher order C-techs
  • Chap3 Computational analysis
    • 3.1 Primary order analysis
    • 3.2 Higer order data analysis
      • 3.2.1 Read mapping consideration
      • 3.2.2 Analytical Pipelines
        • GITAR Pipeline
        • HiC-Pro Pipeline
      • 3.2.3 TAD calling algorithms
    • 3.3 3D structure
  • Chap4 RNA-genome interaction
    • 4.1 Experimental Methods
    • 4.2 Computational Analysis
  • Chap5 Integrative Data Visualization Tools
    • 5.1 GIVE
    • 5.2 HiGlass
  • Chap6 4DN Project
  • Appendix
    • Homework
    • Student's presentation
      • A Brief Introduction to Machine Learning
      • Precision medicine
      • CHIP-Seq
Powered by GitBook
On this page
  • Image based
  • 2.1.1 Visualize DNA & RNA
  • 2.1.2 Ultra structure and 3D organization
  • 2.1.3 Visualizing spatial and dynamic 3D nuclear organization
  1. Chap2 experiment tools for exploring genome interaction

2.1 Image based

PreviousChap2 experiment tools for exploring genome interactionNext2.2 Primary order

Last updated 6 years ago

Image based

  1. Visualize DNA & RNA

    1.1. FISH

    1.2. CRISPR imaging

    1.3. Tagged RNA sequence

  2. Ultra structure and 3D organization

    2.1. EM based methods

    2.2. X-ray based methods

  3. A Visualizing spatial and dynamic 3D nuclear organization

Before the advent of C-technologies, the predominant method to study 3D genome organization was image-based methods.

2.1.1 Visualize DNA & RNA

FISH

FISH (fluorescence in situ hybridization) measured the spatial distances between two or more loci by hybridizing flurescently labeled probes to DNA after fixation, and then visualize the labels under light microscopy.

Limitations:

  • A large number of cells are need to get confident conclusion

  • Resolution is limitted by the diffraction limit of light sources and the size of the probes (like 40kb)

  • Limitted fluorescent labels

Improvements:

CRISPR imaging

Tagged RNA sequence

2.1.2 Ultra structure and 3D organization

EM based methods

X-ray based methods

Super-resolution imaging

2.1.3 Visualizing spatial and dynamic 3D nuclear organization

  • Wide-field / confocal / multiphoton----Live and fixed cells: diffraction limited(>250nm).

  • super resolution imaging tools mentioned in preview section.

Super-resolution microscopy

Short oligonucleotide-based probes

Multiplexed FISH method (more than 30 genomic loci at a time)

HIPMap: High-throughput FISH

seqFISH: profile expressions of hundreds of genes at the single-cell level in situ .

Efforts to label and image endogenous genomic elements in live cells have benefited from the series of modular proteins with specific DNA recognition; like those methods developed for gene editing (zinc-finger modules, TALEs and CRISPR (clustered regularly interspaced short palindromic repeat)-Cas (CRISPR-associated) system), these modules can guide the fluorescence signal to a specific sequence within the complex genome. Comprehensive review can be referred.

Live-cell imaging of mRNA yields important insights into gene expression, transcriptional events. RNA hairpin sequences are inserted in tandem into a gene of interest to tag it, and coexpression of a fluorescent coat protein allows for mRNA detection .

An electron microscope is a microscope that uses a beam of accelerated electrons as a source of illumination. As the wavelength of an electron can be up to 100,000 times shorter than that of visible light photons, electron microscopes have a higher resolving power than light microscopes and can reveal the structure of smaller objects.Transmission electron microscopy, combined with tomographic techniques, has been used for decades to generate 3D representations of biological structures, with resolutions down to the nanometer range.

TEM, serial-section EM tomography----reconstruction of large 3D volumes (250 to > 500 nm thick)

Multi-color EM , ChromEMT ----Local ultrastructure and global 3D organization

soft X-ray tomography (SXT)----image chromatin organization, distribution, and biophysical properties during neurogenesis. Mesoscale resolution (20–50 nm) in intact, unprocessed cells .

Super-resolution microscopy (resolution up to 20nm) has overcome the diffraction limit in common light microscopy (resolution up to 200~300 nm) and enabled visualization of previously invisible molecular details in biological systems. Mainstream technologies are based on two principles ():

Overcome the diffraction limit by accompanying a focused excitation beam with a spatially patterned “depletion” beam, typically in a donut shape, which serves to counteract excitation through either stimulated emission (STED) or other types of fluorescence transitions, such as photoswitching (RESOLFT) .

The second category of methods achieves the separation of molecules by stochastically turning on individual molecules within the diffraction limited volume at different time points, including stochastic optical reconstruction microscopy (STORM) and (fluorescence) photoactivated localization microscopy PALM

Super-resolution microscopy ----Super-resolution imaging of nuclear organization in live and fixed cells (circa 10–20 nm)

[1]
[2]
[3]
[4]
[5]
[6]
[7]
[8]
[9]
[10]
[11]
[12]
[13]
review from Xiaowei Zhuang's lab
[14]
[15]
[16]
[17]
[18]