Application of Lacunarity for Quantification of Single Molecule Localization Microscopy Images

The quantitative analysis of datasets achieved by single molecule localization microscopy is vital for studying the structure of subcellular organizations. Cluster analysis has emerged as a multi-faceted tool in the structural analysis of localization datasets. However, the results it produces greatly depend on the set parameters, and the process can be computationally intensive. Here we present a new approach for structural analysis using lacunarity. Unlike cluster analysis, lacunarity can be calculated quickly while providing definitive information about the structure of the localizations. Using simulated data, we demonstrate how lacunarity results can be interpreted. We use these interpretations to compare our lacunarity analysis with our previous cluster analysis-based results in the field of DNA repair, showing the new algorithm’s efficiency

Quantitative dSTORM superresolution microscopy

Localization based superresolution technique provides the highest spatial resolution in optical microscopy. The final image is formed by the precise localization of individual fluorescent dyes, therefore the quantification of the collected data requires special protocols, algorithms and validation processes. The effects of labelling density and structured background on the final image quality were studied theoretically using the TestSTORM simulator. It was shown that system parameters affect the morphology of the final reconstructed image in different ways and the accuracy of the imaging can be determined. Although theoretical studies help in the optimization procedure, the quantification of experimental data raises additional issues, since the ground truth data is unknown. Localization precision, linker length, sample drift and labelling density are the major factors that make quantitative data analysis difficult. Two examples (geometrical evaluation of sarcomere structures and counting the γH2AX molecules in DNA damage induced repair foci) have been presented to demonstrate the efficiency of quantitative evaluation experimentally

mmSTORM: Multimodal localization based super-resolution microscopy

Super-resolution localization microscopy provides a powerful tool to study biochemical mechanisms at single molecule level. Although the lateral position of the fluorescent dye molecules can be determined routinely with high precision, measurement of other modalities such as 3D and multicolor without the degradation of the original super-resolved image is still in the focus. In this paper a dual-objective multimodal single molecule localization microscopy (SMLM) technique has been developed, optimized and tested. The proposed optical arrangement can be implemented onto a conventional inverted microscope without serious system modification. The performance of the method was tested using fluorescence beads, F-actin filaments and sarcomere structures. It was shown that the proposed imaging method does not degrade the image quality of the original SMLM 2D image but could provide information on the axial position or emission spectra of the dye molecules

Phase separated ribosome-nascent chain complexes in genotoxic stress response

Assemblysomes are EDTA- and RNase-resistant ribonucleoprotein (RNP) complexes of paused ribosomes with protruding nascent polypeptide chains. They have been described in yeast and human cells for the proteasome subunit Rpt1, and the disordered N-terminal part of the nascent chain was found to be indispensable for the accumulation of the Rpt1-RNP into assemblysomes. Motivated by this, to find other assemblysome-associated RNPs we used bioinformatics to rank subunits of Saccharomyces cerevisiae protein complexes according to their N-terminal disorder propensity. The results revealed that gene products involved in DNA repair are enriched among the top candidates. The Sgs1 DNA helicase was chosen for experimental validation. We found that indeed nascent chains of Sgs1 form EDTA-resistant RNP condensates, assemblysomes by definition. Moreover, upon exposure to UV, SGS1 mRNA shifted from assemblysomes to polysomes, suggesting that external stimuli are regulators of assemblysome dynamics. We extended our studies to human cell lines. The BLM helicase, ortholog of yeast Sgs1, was identified upon sequencing assemblysome-associated RNAs from the MCF7 human breast cancer cell line, and mRNAs encoding DNA repair proteins were overall enriched. Using the radiation-resistant A549 cell line, we observed by transmission electron microscopy that 1,6-hexanediol, an agent known to disrupt phase-separated condensates, depletes ring ribosome structures compatible with assemblysomes from the cytoplasm of cells and makes the cells more sensitive to X-ray treatment. Taken together these findings suggest that assemblysomes may be a component of the DNA damage response from yeast to human

Quantitative dSTORM superresolution microscopy

Localization based superresolution technique provides the highest spatial resolution in optical microscopy. The final image is formed by the precise localization of individual fluorescent dyes, therefore the quantification of the collected data requires special protocols, algorithms and validation processes. The effects of labelling density and structured background on the final image quality were studied theoretically using the TestSTORM simulator. It was shown that system parameters affect the morphology of the final reconstructed image in different ways and the accuracy of the imaging can be determined. Although theoretical studies help in the optimization procedure, the quantification of experimental data raises additional issues, since the ground truth data is unknown. Localization precision, linker length, sample drift and labelling density are the major factors that make quantitative data analysis difficult. Two examples (geometrical evaluation of sarcomere structures and counting the γH2AX molecules in DNA damage induced repair foci) have been presented to demonstrate the efficiency of quantitative evaluation experimentally

Application of Lacunarity for Quantification of Single Molecule Localization Microscopy Images

The quantitative analysis of datasets achieved by single molecule localization microscopy is vital for studying the structure of subcellular organizations. Cluster analysis has emerged as a multi-faceted tool in the structural analysis of localization datasets. However, the results it produces greatly depend on the set parameters, and the process can be computationally intensive. Here we present a new approach for structural analysis using lacunarity. Unlike cluster analysis, lacunarity can be calculated quickly while providing definitive information about the structure of the localizations. Using simulated data, we demonstrate how lacunarity results can be interpreted. We use these interpretations to compare our lacunarity analysis with our previous cluster analysis-based results in the field of DNA repair, showing the new algorithm’s efficiency

Phase separated ribosome-nascent chain complexes in genotoxic stress response

Assemblysomes are EDTA- and RNase-resistant ribonucleoprotein (RNP) complexes of paused ribosomes with protruding nascent polypeptide chains. They have been described in yeast and human cells for the proteasome subunit Rpt1, and the disordered N-terminal part of the nascent chain was found to be indispensable for the accumulation of the Rpt1-RNP into assemblysomes. Motivated by this, to find other assemblysome-associated RNPs we used bioinformatics to rank subunits of Saccharomyces cerevisiae protein complexes according to their N-terminal disorder propensity. The results revealed that gene products involved in DNA repair are enriched among the top candidates. The Sgs1 DNA helicase was chosen for experimental validation. We found that indeed nascent chains of Sgs1 form EDTA-resistant RNP condensates, assemblysomes by definition. Moreover, upon exposure to UV, SGS1 mRNA shifted from assemblysomes to polysomes, suggesting that external stimuli are regulators of assemblysome dynamics. We extended our studies to human cell lines. The BLM helicase, ortholog of yeast Sgs1, was identified upon sequencing assemblysome-associated RNAs from the MCF7 human breast cancer cell line, and mRNAs encoding DNA repair proteins were overall enriched. Using the radiation-resistant A549 cell line, we observed by transmission electron microscopy that 1,6-hexanediol, an agent known to disrupt phase-separated condensates, depletes ring ribosome structures compatible with assemblysomes from the cytoplasm of cells and makes the cells more sensitive to X-ray treatment. Taken together these findings suggest that assemblysomes may be a component of the DNA damage response from yeast to human