Imaging the Genome
is unfortunate because cells, includ- ing genomic structure, are highly organized at
the nanoscale, and
structure oſten informs function. To be able to visualize structures well below the diffraction limit using light microscopy, a variety of super- resolution techniques have been devel- oped [5–10]. Tese techniques have already made such a large impact on the field that they were the subject of the Nobel Prize in Chemistry in 2014 [11–13]. One such method, single- molecule localization microscopy (SMLM) [6], has proven to be a useful tool for imaging the genome at super resolution [14]. In SMLM, a diffraction-limited
Figure 1: Chromosome conformation capture technology. (A) DNA-DNA proximity ligation assay of intact nuclei used during in situ Hi-C analysis. (B) Features capable of being revealed by Hi-C maps and corresponding cartoon representations, including subcompartments, domains, and loops. While Hi-C mapping can identify the presence of these features, exact spatial orientation is ambiguous. Figure adapted with permission from its original version [33].
they are based on ensemble data collection. In contrast, imag- ing provides data on individual cells resulting in an actual 3D representation of the conformation because distances within a genomic region are being measured directly rather than inferred, based on sequencing results. Imaging is also capa- ble of showing relationships between multiple chromosomes within a single experiment at high resolution, which could be missed by 3C techniques. However, imaging is limited in comparison to 3C in the number of cells being analyzed, that is, tens or thousands of cells in comparison to millions. Te advantage of imaging is that it provides single cell as opposed to ensemble data, enabling visualization of cellular context.
A Brief Introduction to Single-Molecule Imaging Biological systems exist across a wide range of sizes. Light
microscopy, particularly fluorescence microscopy, has been a useful tool for visualizing biological organization. How- ever, traditional approaches are unable to resolve features below about 200 nm due to Abbe’s diffraction limit [4]. Tis
20
object is densely labeled with photo- switchable fluorescent molecules. In conventional fluorescence micros- copy, the underlying structure is obscured due to Abbe’s diffraction limit. With SMLM, in the presence of the correct photochemical condi- tions, these molecules are turned off and then turned back on in a stochas- tic manner. Tis allows the position of each molecule to be recorded dur- ing image acquisition. At the end of an acquisition, all the center points are then used to reconstruct a final super-resolution image (Figure 2). Extending this method further, it is possible to obtain 3D information by a variety of means. A common approach to 3D SMLM is the use of a cylindrical lens in the detection path, which generates an elongated point- spread function (PSF) in either X or
Y, depending on the positioning of a molecule in Z [15]. Tis astigmatic approach is limited to using total internal reflection fluorescence (TIRF) or highly inclined and laminated optical sheet (HILO) illumination, and relies on point-spread-func- tion engineering, restricting the depth of imaging from the cover slip to less than 5–10 μm. An alternative to astigmatic imaging is biplane imaging
[16], where a fiſty-fiſty beam splitter is placed in the detection path. Tis divides the light into two paths to be imaged on different parts of the camera. Te second path is longer and uses a larger focal length lens to image onto the camera. Tis effectively separates the focal length of the two paths in Z (approximately 600 nm in practice), allowing two Z planes to be imaged simultaneously for the same field of view (FOV). Furthermore, prior to imaging, a calibration table is created based on the PSF of the fluorophores within each plane at known Z positions to allow the axial position of each molecule to be determined during imaging. With this approach, a single focal-plane acquisition provides axial information of 1 μm for
www.microscopy-today.com • 2020 November
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