For centuries, light microscopy has greatly facilitated our understanding of how cells function. In fact, entire fields of biology have emerged from images acquired under light microscopes. Indeed, one major element that makes light microscopy so powerful in biological research is the development of various staining methods that permit the labeling of specific molecules and cells. For example, fluorescence in situ hybridization (FISH) detects DNA and RNA molecules with specific sequences, whereas immunofluorescence labels and fluorescent proteins allow the imaging of particular proteins in cells
The unrivaled combination of molecule-specific contrast and live-cell imaging capability makes fluorescence microscopy the most popular imaging modality in cell biology. However, the application of fluorescence microscopy to many areas of biology is still hindered by its moderate resolution of several hundred nanometers. This resolution is approximately the size of an organelle and thus is inadequate for dissecting the inner architecture of many subcellular structures.
The resolution for optical microscopy is limited by the diffraction, or the “spreading out,” of the light wave when it passes through a small aperture or is focused to a tiny spot. Because this property is intrinsic to all waves, breaking the diffraction barrier of light microscopy has been deemed impossible for a long time. However, such limitations have not deterred a small group of scientists from pursuing “super-resolution” fluorescence microscopy that breaks through this seemingly impenetrable barrier.
Super-resolution microscopy is a series of techniques in optical microscopy that allow such images to have resolutions higher than those imposed by the diffraction limit, which is due to the diffraction of light. Super-resolution imaging techniques rely on the near-field (photon-tunneling microscopy as well as those that utilize the Pendry Superlens and near field scanning optical microscopy) or on the far-field.
Among techniques that rely on the latter are those that improve the resolution only modestly (up to about a factor of two) beyond the diffraction-limit, such as confocal microscopy with closed pinhole or aided by computational methods such as deconvolution or detector-based pixel reassignment (e.g. re-scan microscopy, pixel reassignment), the 4Pi microscope, and structured-illumination microscopy technologies such as SIM and SMI.
Superresolution Method Poised to Improve Gene Function Understanding
An interdisciplinary team from the Centre for Genomic Regulation (CRG) and the Institute for Research in Biomedicine (IRB Barcelona) has developed an imaging technique that captures the structure of the human genome to reveal how individual genes fold at the nucleosome level — the fundamental units constituting the genome’s three-dimensional architecture. The technique integrates superresolution imaging with advanced computational modeling.
According to the researchers, the method allowed them to image the structure of the human genome at unprecedented resolution. They believe the technique could have a long-term impact on scientific discovery.
Scientists used the technology, called Modeling immuno-OligoSTORM (MiOS), to create and virtually navigate 3D models of genes. Since almost every human disease has some basis in genes, the ability to visualize how genes work could lead scientists to a better understanding of how genes affect the health of the human body. The developers of MiOS hypothesized that taking superresolution microscopy and merging it with advanced computational tools could be a way to image genes at the level of detail necessary to study their shape and function, so as to fully understand their function and regulation.
MiOS showed the distribution of nucleosomes within specific genes in superresolution, through the simultaneous visualization of DNA and histones. It integrated this information with restraint-based and coarse-grained modeling approaches. It allowed quantitative modeling of genes with nucleosome resolution and provides information about chromatin accessibility for regulatory factors such as RNA polymerase II.
The researchers used MiOS to explore intercellular variability, transcriptional-dependent gene conformation, and folding of housekeeping and pluripotency-related genes in human pluripotent and differentiated cells. MiOS demonstrated a high degree of resolution and data integration, revealing structures and details in genes that are not captured using conventional techniques.
According to researcher Juan Pablo Arcon, the method provided a picture, or movie, of the 3D shape of genes at resolutions beyond the size of nucleosomes, reaching the scales that are needed to understand in detail the interaction between chromatin and other cell factors.
“We show that MiOS provides unprecedented detail by helping researchers virtually navigate inside genes, revealing how they are organized at a completely new scale,” researcher Vicky Neguembor said. “It is like upgrading from the Hubble Space Telescope to the James Webb, but instead of seeing distant stars we’ll be exploring the farthest reaches inside a human nucleus.”
In the future, observations on genetic information obtained through MiOS could be used to catalog variations in the shape of genes that cause disease, for example. MiOS could also be used to test drugs that may be able to treat a disease by changing the shape of an aberrant gene.
The researchers intend to develop MiOS further, adding additional functionality that can, for example, detect how transcription factors (i.e., proteins involved in the process of transcribing DNA into RNA) bind to DNA. The research was published in Nature: Structural & Molecular Biology (www.doi.org/10.1038/s41594-022-00839-y).
References and Resources also include: