Super-resolution microscopy overcomes the diffraction limit of conventional light microscopy by at least a factor of two, providing a resolution of 100 nm or smaller. Diffraction is a manifestation of the wave properties of light. In a typical light microscope operating in the visible spectral range (400-700 nm) (see Figure below), diffraction determines the smallest focal volume that light can be focused into.The diffraction limit of light microscopes, known as the point spread function (PSF), limits resolution to ~250 nm in the X-Y image plane and ~500 nm along the Z optical axis . In conventional light microscopes such as confocal or TIRF (Total Internal Reflection Fluorescence), structural features lying closer to each other than the PSF length scale cannot be resolved. This includes cellular structures that contribute to cell motility, force generation and mechanosensing such as actin filaments, microtubules and focal adhesion complexes.This limitation has however been over come with the development of various super resolution microscopy methods.
Binding activatable localization microscopy (BALM) enables a resolution of ~30nm. This imaging technique was developed based on a similar principle as photoactivatable localization microscopy (PALM) and stochastic optical reconstruction microscopy (STORM). The illumination of a sparse set of fluorophores enables the detection of single molecules, and the repetitive capturing of such sparse sets of fluorophores on a total internal fluorescence (TIRF) microscopy is used to reconstruct superresolution images.In BALM, fluorophores are “switched on” when bound to the target structure, for example, DNA-binding dyes that show a strong fluorescence enhancement when bound to dsDNA, such as YOYO-1. YOYO-1 fluoresces 800-1000 times more when compared to free YOYO-1 molecules.
The nuclear architecture is further determined by a non-random, three-dimensional positioning of the chromosomes into distinct chromosomes territories. This spatial organization of chromatin contributes further to the regulation of gene expression. This has been shown in studies exploring the radial alignment of chromosomes, where gene-active chromosomes were shown to localize near the center of the nucleus and gene-inactive chromosomes at the nuclear periphery. Additionally, studies using chromosomal capture assays have demonstrated the presence of chromosome neighborhoods, such as interchromosomal compartments (ICs), which house the transcription machinery. These ICs function as regions for gene co-regulation, where gene-rich regions of neighboring chromosomes form loops that intermingle, thereby allowing transcriptional machinery to be shared by neighboring chromosomes.