Super-resolution imaging of a 2.5 kb non-repetitive DNA in situ in the nuclear genome using molecular beacon probes

Abstract

High-resolution visualization of short non-repetitive DNA in situ in the nuclear genome is essential for studying looping interactions and chromatin organization in single cells. Recent advances in fluorescence in situ hybridization (FISH) using Oligopaint probes have enabled super-resolution imaging of genomic domains with a resolution limit of 4.9 kb. To target shorter elements, we developed a simple FISH method that uses molecular beacon (MB) probes to facilitate the probe-target binding, while minimizing non-specific fluorescence. We used three-dimensional stochastic optical reconstruction microscopy (3D-STORM) with optimized imaging conditions to efficiently distinguish sparsely distributed Alexa-647 from background cellular autofluorescence. Utilizing 3D-STORM and only 29–34 individual MB probes, we observed 3D fine-scale nanostructures of 2.5 kb integrated or endogenous unique DNA in situ in human or mouse genome, respectively. We demonstrated our MB-based FISH method was capable of visualizing the so far shortest non-repetitive genomic sequence in 3D at super-resolution.DOI: http://dx.doi.org/10.7554/eLife.21660.001

Lipid rafts increase to facilitate ectoderm lineage specification of differentiating embryonic stem cells

“…Although conventional fluorescence microscopy and flow cytometry demonstrated that the abundance of cell-surface rafts increased during RA-induced differentiation ( Figure 1 ), they failed to reveal the detailed features of these nanoscopic structures. In this regard, we sought to STORM that provides better spatial resolution, e.g lateral resolution of 22 nm in our lab [38]. STORM breaks optical diffraction limit via stochastically activating switchable fluorophores like Alexa Fluor 647 (AF647) at separate times, and the final super-resolution images are reconstructed with the measured positions of these individual fluorophores [7-9].…”

“…A STORM system based on an inverted optical microscope (IX-71, Olympus) with a 100x oil immersion objective lens (UplanSApo, N.A. 1.40, Olympus) was used for the nanoimaging as previously described [38]. A 405 nm laser (CUBE 405–100C; Coherent) was used for photoactivation and a 641 nm laser (CUBE 640–100C; Coherent) was used to excite fluorescence and deactivate Alexa-647 to the dark state.…”

“…The STORM imaging data were analyzed in a similar manner as previously described [38]. Briefly, the fluorescence images of each fluorophore were preprocessed using a band-pass filter to reduce the background noise, and then fitted to a Gaussian function where the position of the fluorophore in the x-y plane was directly read out.…”

“…In the reconstructed image, each localization was represented as a spot whose diameter matched with the localization precision, i.e. 22 nm [38]. Only those clusters containing >5 localizations were determined as raft[10] then painted in the super-resolution images and subjected to further analysis.…”

Chromatin imaging and new technologies for imaging the nucleome

“…The molecular beacon resumes fluorescence upon binding to its target. Ni et al () demonstrated the utilization of mbFISH and STORM by imaging a 2.5‐kb nonrepetitive locus. Other techniques, such as fluorescence resonance energy transfer (Auer, Strauss, Schlichthaerle, & Jungmann, ) have also been utilized to eliminate background fluorescence in DNA PAINT.…”