Table 3 Imaging technologies currently in use or in development in the 4DN Network
From: The 4D nucleome project
Application | Method (PMID) | Label/stain/dye | Feature |
---|---|---|---|
Visualizing DNA and RNA sequences | Fl-labelled oligo and BAC probes | Fixed cells, single- and multiple-target DNA and RNA detection | |
HIPMap82 | Fl-labelled probes | High-throughput FISH and automated microscopy in fixed cells | |
Fl-labelled dCas9 and guide RNAs (gRNAs) to label genomic loci; gRNAs can contain a stem loop bound by Fl-labelled coat proteins | Live and fixed cells–gRNAs against repetitive targets and/or collections of gRNAs that cover unique sequences | ||
Track first, identify later84 | Barcoded Fl-labelled oligos and Fl-dCas9 | Combination of SeqFISH and Cas9 live-cell imaging | |
Fl-fusions that bind tagged RNA sequences | Live and fixed cells: transcription | ||
Visualized by Fl-labelled ParB-, lac- or tet-repressor proteins | Live and fixed cells: genomic tags | ||
High resolution ultrastructure and 3D organization | Transmission EM, SBEM, multiple-tilt89 and serial-section EM tomography90 | Heavy metals, colloidal nanogold, inorganic and organic probes and tags, ChromEM, Alexa 633, Fluoronanogold, Time-STAMP-YFP-MiniSOG | High resolution ultrastructure of macromolecules in situ ranging from 2D transverse projections through 70–250-nm sections to reconstruction of large 3D volumes (250 to >500 nm thick) |
Orthogonal-correlated light and EM probes and DAB conjugates | Local ultrastructure and global 3D organization of chromatin as a continuum at nucleosome resolutions and megabase scales | ||
X-ray tomography41 | Quantitative LAC-generated contrast that reflects the bioorganic composition of unlabelled DNA, RNA and proteins | Mesoscale resolution (20–50 nm) in intact, unprocessed cells | |
Correlated SIM and X-ray tomography42 | Fl-labelled probes | Visualize ultrastructure and interactions of selectively labelled chromatin and protein simultaneously | |
Visualizing spatial and dynamic 3D nuclear organization | Wide-field, confocal/multiphoton | Fl probes: Fl protein, SNAP and Halo genomic tags, Alexa and Cy dyes, atto488, Suntag, SH2, Quantum dots, Janelia fluor dyes101 | Live and fixed cells: diffraction limited (>250 nm) |
Super resolution: 3D SIM, PALM, STORM92, STED, adaptive optics93, tcPALM28, SAIM94 | Fl probes, photo-activatable and photo-switchable proteins; caged organic fluorophores101 and organic fluorophores in thiol buffer coupled to SNAP, Halo, nanobodies or FISH oligos | Super-resolution imaging of nuclear organization in live and fixed cells (circa 10–20 nm) | |
Fl-labelled probes | Live cell: 3D dynamics and nuclear organization | ||
Fl-labelled and photo-activatable probes, ArrayG | Live cell: binding and search dynamics: residence time, search time, search mechanism |