Superresolution intrinsic fluorescence imaging of chromatin utilizing native, unmodified nucleic acids for contrast

Abstract

Visualizing the nanoscale intracellular structures formed by nucleic acids, such as chromatin, in nonperturbed, structurally and dynamically complex cellular systems, will help expand our understanding of biological processes and open the next frontier for biological discovery. Traditional superresolution techniques to visualize subdiffractional macromolecular structures formed by nucleic acids require exogenous labels that may perturb cell function and change the very molecular processes they intend to study, especially at the extremely high label densities required for superresolution. However, despite tremendous interest and demonstrated need, label-free optical superresolution imaging of nucleotide topology under native nonperturbing conditions has never been possible. Here we investigate a photoswitching process of native nucleotides and present the demonstration of subdiffraction-resolution imaging of cellular structures using intrinsic contrast from unmodified DNA based on the principle of single-molecule photon localization microscopy (PLM). Using DNA-PLM, we achieved nanoscopic imaging of interphase nuclei and mitotic chromosomes, allowing a quantitative analysis of the DNA occupancy level and a subdiffractional analysis of the chromosomal organization. This study may pave a new way for label-free superresolution nanoscopic imaging of macromolecular structures with nucleotide topologies and could contribute to the development of new DNA-based contrast agents for superresolution imaging.superresolution fluorescence microscopy | label-free imaging | nucleic acids | chromatin topology | chromosome A dvances in genomics and molecular biology over the past decades revolutionized our knowledge of biological systems. Despite our expanded understanding of biological interactions, there continues to be a limited understanding of these complex molecular processes in nonperturbed, structurally and dynamically complex cellular systems (1). As such, it is of critical importance to develop methods that allow direct visualization of nanoscale structures where these processes take place in their native states. Recently, superresolution fluorescence microscopy techniques, including stimulated emission depletion microscopy, structured illumination microscopy, and photon localization microscopy (PLM), such as photoactivated localization microscopy and stochastic optical reconstruction microscopy (STORM), have extended the ultimate resolving power of optical microscopy far beyond the diffraction limit (2-6), facilitating access to the organization of cells at the nanoscale by optical means. Although superresolution imaging of biological structures using labeled proteins has been well documented due to a wide range of methodologies that provide desirable labeling properties (7,8), and despite tremendous interest and demonstrated need, there are few nanoscopic methods to image macromolecular structures formed by nucleic acids due to constraints in labeling (9-14). Likewise, the limited techniques that currently exist cannot ...

Measurement of complex DNA damage induction and repair in human cellular systems after exposure to ionizing radiations of varying linear energy transfer (LET)

“…The use of other DNA repairs foci like RAD51, MDC1 for DSBs and XRCC1, for example for non-DSBs maybe can help toward the optimization of detection according to the type of genotoxic stress [58]. In addition, very recent advances in fluorescence microscopy harnessing previously unused fluorescence or DNA autofluorescence by a 532-nm laser excitation [59] may improve the detection of protein distributions especially located in a complex form [60].…”

Multiplexed RNAi therapy against brain tumor-initiating cells via lipopolymeric nanoparticle infusion delays glioblastoma progression

“…Super-Resolution SMLM Analysis. To enhance the optical resolution of subcellular organelles and nanoscale LPNP aggregates further after biological uptake into the cytosol of BTICs, we performed super-resolution imaging of BTICs after LPNP uptake at different time points, using a custom SMLM super-resolution nanoscopy (79,80). To perform SMLM imaging, 5-μL cell suspensions of each sample were mixed with the imaging buffer and deposited at the center of a freshly cleaned glass slide.…”

Stochastic fluorescence switching of nucleic acids under visible light illumination

“…This intrinsic fluorescence has likely been overlooked previously because most photochemical studies of nucleotides were performed in dilute solutions with concentrations ranging between 10-100 µM [15][16][17][18], which is significantly lower than that in nuclei and chromosomes (0.1-1 M) [19][20][21]. More importantly, we further observed the stochastic fluorescence switching of nucleotides under visible illumination, which sets the foundation of using unmodified nucleic acids as endogenous contrast agents for nanoscopic imaging with photon localization microscopy (PLM) [14]. This phenomenon sets the stage for developing new label-free super-resolution optical imaging methods to resolve macromolecular structures with nucleotide topologies.…”

“…up to 10 5 kWcm −2 in STED). In all, this discovery paves a new way to realize label-free optical super-resolution imaging of nucleic acids [14], which may provide an ideal technique to visualize the spatial organization of single or groups of nucleosomes and quantitatively estimates the nucleosome occupancy level of DNA in unstained chromosomes and nuclei. …”

“…It is wellknown that nucleotides fluoresce under UV illumination and have significantly weaker absorption in the visible range. However, we have observed visible light-excited fluorescence of unmodified nucleic acids when their concentration approached the value of physiological conditions [14]. This intrinsic fluorescence has likely been overlooked previously because most photochemical studies of nucleotides were performed in dilute solutions with concentrations ranging between 10-100 µM [15][16][17][18], which is significantly lower than that in nuclei and chromosomes (0.1-1 M) [19][20][21].…”