Table 3: Imaging technologies currently in use or in development in the 4DN Network

From: The 4D nucleome project

ApplicationMethod (PMID)Label/stain/dyeFeature
Visualizing DNA and RNA sequences3D DNA and RNA FISH78, seqFISH79, MERFISH80,81Fl-labelled oligo and BAC probesFixed cells, single- and multiple-target DNA and RNA detection
 HIPMap82Fl-labelled probesHigh-throughput FISH and automated microscopy in fixed cells
 CASFISH31,83, CRISPRainbow29, Cas live-cell imaging30,38Fl-labelled dCas9 and guide RNAs (gRNAs) to label genomic loci; gRNAs can contain a stem loop bound by Fl-labelled coat proteinsLive and fixed cells–gRNAs against repetitive targets and/or collections of gRNAs that cover unique sequences
 Track first, identify later84Barcoded Fl-labelled oligos and Fl-dCas9Combination of SeqFISH and Cas9 live-cell imaging
 MS2, PP7, mSpinach, aptamers85,86Fl-fusions that bind tagged RNA sequencesLive and fixed cells: transcription
 ParS-, LacO- or TetO-labelled genomic loci87,88Visualized by Fl-labelled ParB-, lac- or tet-repressor proteinsLive and fixed cells: genomic tags
High resolution ultrastructure and 3D organizationTransmission EM, SBEM, multiple-tilt89 and serial-section EM tomography90Heavy metals, colloidal nanogold, inorganic and organic probes and tags, ChromEM, Alexa 633, Fluoronanogold, Time-STAMP-YFP-MiniSOGHigh 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)
 Multi-colour EM91, ChromEMT (UD)43Orthogonal-correlated light and EM probes and DAB conjugatesLocal ultrastructure and global 3D organization of chromatin as a continuum at nucleosome resolutions and megabase scales
 X-ray tomography41Quantitative LAC-generated contrast that reflects the bioorganic composition of unlabelled DNA, RNA and proteinsMesoscale resolution (20–50 nm) in intact, unprocessed cells
 Correlated SIM and X-ray tomography42Fl-labelled probesVisualize ultrastructure and interactions of selectively labelled chromatin and protein simultaneously
Visualizing spatial and dynamic 3D nuclear organizationWide-field, confocal/multiphotonFl probes: Fl protein, SNAP and Halo genomic tags, Alexa and Cy dyes, atto488, Suntag, SH2, Quantum dots, Janelia fluor dyes101Live and fixed cells: diffraction limited (>250 nm)
 Super resolution: 3D SIM, PALM, STORM92, STED, adaptive optics93, tcPALM28, SAIM94Fl probes, photo-activatable and photo-switchable proteins; caged organic fluorophores101 and organic fluorophores in thiol buffer coupled to SNAP, Halo, nanobodies or FISH oligosSuper-resolution imaging of nuclear organization in live and fixed cells (circa 10–20 nm)
 LLS, LLS-PAINT95,96Fl-labelled probesLive cell: 3D dynamics and nuclear organization
 Single-molecule imaging and tracking97,98,99,100Fl-labelled and photo-activatable probes, ArrayGLive cell: binding and search dynamics: residence time, search time, search mechanism
  1. BAC, bacterial artificial chromosome; CasFISH, CRISPR-associated protein 9 (Cas9) fluorescence in situ hybridization; CRISPRainbow, multiplexed labelling of genomic loci with dCas9 and engineered sgRNAs; DAB, 3,4-diaminobenzidine; EM, electron microscopy; FISH, fluorescence in situ hybridization; Fl, fluorescent; HIPMap, high-throughput imaging position mapping; LAC, linear absorption coefficient; LLS, lattice light sheet; LLS-PAINT, lattice light sheet point accumulation for imaging in nanoscale topography; MERFISH, multiplexed error-robust fluorescence in situ hybridization; mini-SOG, mini singlet oxygen generator; MS2 and PP7, bacteriophage RNA stem loop motifs; PALM, photo-activated localization microscopy; SAIM, scanning angle interference microscopy; SBEM, serial block face scanning electron microscopy; seqFISH, sequential live and fixed fluorescence in situ hybridization; SIM, structured illumination microscopy; SNAP, a variant of O6-alkylguanine-DNA alkyltransferase that reacts specifically with benzylguanine; STAMP, sequentially timed all-optical mapping photography; STED, stimulated emission depletion; STORM, stochastic optical reconstruction microscopy; tcPALM, time-correlated PALM; UD, under development.