2.1 Image based
Image based
Visualize DNA & RNA
1.1. FISH
1.2. CRISPR imaging
1.3. Tagged RNA sequence
Ultra structure and 3D organization
2.1. EM based methods
2.2. X-ray based methods
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 [1] (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:
Super-resolution microscopy [2]
Short oligonucleotide-based probes [3]
Multiplexed FISH method (more than 30 genomic loci at a time) [4]
HIPMap: High-throughput FISH [5]
seqFISH: profile expressions of hundreds of genes at the single-cell level in situ [6].
CRISPR imaging
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 [7] can be referred.
Tagged RNA sequence
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 [8].
2.1.2 Ultra structure and 3D organization
EM based methods
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.[9]
TEM, serial-section EM tomography----reconstruction of large 3D volumes (250 to > 500 nm thick) [10]
X-ray based methods
soft X-ray tomography (SXT)----image chromatin organization, distribution, and biophysical properties during neurogenesis. Mesoscale resolution (20–50 nm) in intact, unprocessed cells [13].
Super-resolution imaging
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 (review from Xiaowei Zhuang's lab):
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) [14] or other types of fluorescence transitions, such as photoswitching (RESOLFT) [15].
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) [16] and (fluorescence) photoactivated localization microscopy PALM [17]
2.1.3 Visualizing spatial and dynamic 3D nuclear organization
Wide-field / confocal / multiphoton----Live and fixed cells: diffraction limited(>250nm).
Super-resolution microscopy [18]----Super-resolution imaging of nuclear organization in live and fixed cells (circa 10–20 nm)
super resolution imaging tools mentioned in preview section.
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