Imaging the Genome
Figure 5: OligoSTORM imaging examples. (A) Several chromosomes traced in parallel. 8.16 Mbs of chromosome 19 was traced while also tracing chromosomes 3, 5, and 8 in uniform step sizes of 500, 250, and 100 kb, respectively. Both homologs of all chromosomes were captured. This was possible, in part, because imaging was conducted (in 100 nm increments) along the Z axis for up to 4 μm, well within the volumetric capability of the Vutara and sufficient to traverse the depth of PGP1f nuclei. (B) An isosurface rendering of maternal and paternal homologs of chromosome 19; fourteen unique barcodes are shown. (C) OligoSTORM and ensemble Hi-C data can be integrated to produce a 3D model of two homologous genomic regions in a single nucleus via integrative modeling of genomic regions (IMGR). IMGR uses rigid- body fitting and refinement through flexible fitting to achieve genomic resolutions of 10 kb or better for regions that have been imaged in much larger step sizes. Panels A and C adapted with permission from their original versions [14].
be localized. A popular use of this technique relies on DNA (DNA-PAINT [26,27]) barcoded antibodies and their comple- mentary sequences bearing a fluorescent tag. Te DNA probes are designed to be short enough so that during imaging they bind and fall off the DNA barcoded antibody, allowing only bound probes to be imaged and localized. Tis approach is advantageous compared to methods like (d)STORM when it comes to fluorophore selection, as fluorophores with high pho- ton budgets (without regard for their photoswitching capabili- ties) can be used resulting in high localization precision. Te drawback to DNA-PAINT is oſten the imaging time. Because it is reliant on the diffusion of probes, typical imaging times are much longer, with exposure times 5–10 times greater than (d) STORM. However, recent advancement in fluorogenic probes is making this less of an issue [28]. A final key advantage of DNA-PAINT is it allows for easy multiplexing [27] due to the nature of its probes (Figure 3).
Imaging the Genome with Single-Molecule Localization Microscopy A natural extension of multiplexed imaging strategies
like DNA-PAINT is OligoSTORM [14], which is the applica- tion of oligopaint probes to image the genome. Tis technique combines the multiplexing capabilities of PAINT with the speed of (d)STORM imaging, allowing for direct visualiza- tion of chromatin structure in situ. Tis technique has been used successfully to perform multi-chromosome walks. With
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this method, special probes made of DNA sequences that complement a genomic region are designed to label specific genes. Tese probes also bear a region for binding other flu- orophore-labeled DNA strands that serve as imaging probes. Te regions can bind longer DNA sequences for use in Oli- goSTORM imaging, or shorter sequences for Ol igoDNA- PAINT (Figure 4). Te differ- ence in the two options is that OligoSTORM relies on blink- ing of the fluorescent molecule to generate localizations, where the OligoDNA-PAINT local- izations come from the tran- sient binding of DNA on the sample. Having both options available also allows for the same region to be imaged twice if necessary. Aſter developing a set of oligopaint probes, they can be imaged sequentially (Figure 4). Te broader workflow of
these experiments involves the development of a library of many thousands of oligo- nucleotides and generation
of OligoSTORM probes that are labeled on a sample. Fluid- ics are then used to label the probes individually so that each probe can be imaged by single-molecule localization. From there, data are driſt corrected and analyzed so that a final image can be assembled for each genomic walk (Figure 4). Tis workflow has achieved localization precisions of ∼20 nm in XY and 50 nm in Z, obtaining subsequent optical resolu- tions of ∼25 nm in XY and 110 nm in Z using Bruker’s Vutara SMLM platform. Tis methodology can provide a clear picture of analogous regions on multiple chromosomes, the relation- ships between sections of a single chromosome, and even finer details, such as individual loops (Figure 5). Analysis of these datasets enables generation of in situ Hi-C maps that exhibit strong correspondence to ensemble Hi-C maps, indicating the validity of image-based measurements and the overall capabil- ity of OligoSTORM to elucidate the organization of chromo- somes. Furthermore, OligoSTORM data and analysis can be combined with ensemble Hi-C data to generate 3D genomic models at resolutions of 10 kb despite being imaged at much larger step sizes in a technique known as integrative modeling of genomic regions (IMGR) (Figure 5).
A Genomic Imaging Solution Establishment of a robust acquisition and analysis pipeline
complete with integrated fluidics is essential for performing large genomic walks. Bruker’s Vutara SMLM imaging platform combined with integrated fluidics makes sequential imaging
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