Patterned illumination single molecule localization microscopy (piSMLM): user defined blinking regions of interest.

Single molecule localization microscopy (SMLM) has been established as an important super-resolution technique for studying subcellular structures with a resolution down to a lateral scale of 10 nm. Usually samples are illuminated with a Gaussian shaped beam and consequently insufficient irradiance on the periphery of the illuminated region leads to artifacts in the reconstructed image which degrades image quality. We present a newly developed patterned illumination SMLM (piSMLM) to overcome the problem of uneven illumination by computer-generated holography. By utilizing a phase-only spatial light modulator (SLM) in combination with a modified Gerchberg-Saxton algorithm, a user-defined pattern with homogeneous and nearly speckle-free illumination is obtained. Our experimental results show that irradiance 1 to 5 kW/cm2 was achieved by using a laser with an output power of 200 mW in a region of 2000 µm2 to 500 µm2, respectively. Higher irradiance of up to 20 kW/cm2 can be reached by simply reducing the size of the region of interest (ROI). To demonstrate the application of the piSMLM, nuclear structures were imaged based on fluctuation binding-activated localization microscopy (fBALM). The super-resolution fBALM images revealed nuclear structures at a nanometer scale.

Super-resolution localization microscopy of radiation-induced histone H2AX-phosphorylation in relation to H3K9-trimethylation in HeLa cells.

Ionizing radiation (IR)-induced damage confers functional and conformational changes to nuclear chromatin associated with DNA single and double strand breaks. This leads to the activation of complex DNA repair machineries that aim to preserve the integrity of the DNA molecule. Since hetero- and euchromatin are differentially accessible to DNA repair pathways, local chromatin re-arrangements and structural changes are among the consequences of an activated DNA damage response. Using super-resolution localization microscopy (SRLM), we investigated the X-ray-induced repositioning of γ-H2AX and histone H3K9me3 heterochromatin marks in the nuclei of HeLa cells. Aliquots of cells exposed to different IR doses (0.5, 1 and 2 Gy) were fixed at certain repair times for SRLM imaging. The number and size of nano-scale γ-H2AX molecule signal clusters detected increased with rising irradiation doses, with the number and size being the highest 0.5 h after irradiation. With growing repair time both the number and size of γ-H2AX nano-clusters decreased. Eight hours after irradiation, the number of clusters reached control levels, in agreement with the disappearance of most IR-induced foci seen by conventional microscopy. SRLM investigation of heterochromatin marks in spatial relation to γ-H2AX clusters showed that on average the heterochromatin density was high in the vicinity of γ-H2AX, which is in agreement with the observation that DSBs seem to relocate to the surface of heterochromatin clusters for DNA repair. The data demonstrate the potential of pointillist images obtained by SRLM for quantitative investigations of chromatin conformation changes and repair-protein recruitment on the nanoscale as measures for a radiation response.

Localization Microscopy Analyses of MRE11 Clusters in 3D-Conserved Cell Nuclei of Different Cell Lines.

In radiation biophysics, it is a subject of nowadays research to investigate DNA strand break repair in detail after damage induction by ionizing radiation. It is a subject of debate as to what makes up the cell's decision to use a certain repair pathway and how the repair machinery recruited in repair foci is spatially and temporarily organized. Single-molecule localization microscopy (SMLM) allows super-resolution analysis by precise localization of single fluorescent molecule tags, resulting in nuclear structure analysis with a spatial resolution in the 10 nm regime. Here, we used SMLM to study MRE11 foci. MRE11 is one of three proteins involved in the MRN-complex (MRE11-RAD50-NBS1 complex), a prominent DNA strand resection and broken end bridging component involved in homologous recombination repair (HRR) and alternative non-homologous end joining (a-NHEJ). We analyzed the spatial arrangements of antibody-labelled MRE11 proteins in the nuclei of a breast cancer and a skin fibroblast cell line along a time-course of repair (up to 48 h) after irradiation with a dose of 2 Gy. Different kinetics for cluster formation and relaxation were determined. Changes in the internal nano-scaled structure of the clusters were quantified and compared between the two cell types. The results indicate a cell type-dependent DNA damage response concerning MRE11 recruitment and cluster formation. The MRE11 data were compared to H2AX phosphorylation detected by γH2AX molecule distribution. These data suggested modulations of MRE11 signal frequencies that were not directly correlated to DNA damage induction. The application of SMLM in radiation biophysics offers new possibilities to investigate spatial foci organization after DNA damaging and during subsequent repair.

Combining Low Temperature Fluorescence DNA-Hybridization, Immunostaining, and Super-Resolution Localization Microscopy for Nano-Structure Analysis of ALU Elements and Their Influence on Chromatin Structure.

Immunostaining and fluorescence in situ hybridization (FISH) are well established methods for specific labelling of chromatin in the cell nucleus. COMBO-FISH (combinatorial oligonucleotide fluorescence in situ hybridization) is a FISH method using computer designed oligonucleotide probes specifically co-localizing at given target sites. In combination with super resolution microscopy which achieves spatial resolution far beyond the Abbe Limit, it allows new insights into the nano-scaled structure and organization of the chromatin of the nucleus. To avoid nano-structural changes of the chromatin, the COMBO-FISH labelling protocol was optimized omitting heat treatment for denaturation of the target. As an example, this protocol was applied to ALU elements-dispersed short stretches of DNA which appear in different kinds in large numbers in primate genomes. These ALU elements seem to be involved in gene regulation, genomic diversity, disease induction, DNA repair, etc. By computer search, we developed a unique COMBO-FISH probe which specifically binds to ALU consensus elements and combined this DNA-DNA labelling procedure with heterochromatin immunostainings in formaldehyde-fixed cell specimens. By localization microscopy, the chromatin network-like arrangements of ALU oligonucleotide repeats and heterochromatin antibody labelling sites were simultaneously visualized and quantified. This novel approach which simultaneously combines COMBO-FISH and immunostaining was applied to chromatin analysis on the nanoscale after low-linear-energy-transfer (LET) radiation exposure at different doses. Dose-correlated curves were obtained from the amount of ALU representing signals, and the chromatin re-arrangements during DNA repair after irradiation were quantitatively studied on the nano-scale. Beyond applications in radiation research, the labelling strategy of immunostaining and COMBO-FISH with localization microscopy will also offer new potentials for analyses of subcellular elements in combination with other specific chromatin targets.

Single-Molecule Localization Microscopy allows for the analysis of cancer metastasis-specific miRNA distribution on the nanoscale.

We describe a novel approach for the detection of small non-coding RNAs in single cells by Single-Molecule Localization Microscopy (SMLM). We used a modified SMLM-setup and applied this instrument in a first proof-of-principle concept to human cancer cell lines. Our method is able to visualize single microRNA (miR)-molecules in fixed cells with a localization accuracy of 10-15 nm, and is able to quantify and analyse clustering and localization in particular subcellular sites, including exosomes. We compared the metastasis-site derived (SW620) and primary site derived (SW480) human colorectal cancer (CRC) cell lines, and (as a proof of principle) evaluated the metastasis relevant miR-31 as a first example. We observed that the subcellular distribution of miR-31 molecules in both cell lines was very heterogeneous with the largest subpopulation of optically acquired weakly metastatic cells characterized by a low number of miR-31 molecules, as opposed to a significantly higher number in the majority of the highly metastatic cells. Furthermore, the highly metastatic cells had significantly more miR-31-molecules in the extracellular space, which were visualized to co-localize with exosomes in significantly higher numbers. From this study, we conclude that miRs are not only aberrantly expressed and regulated, but also differentially compartmentalized in cells with different metastatic potential. Taken together, this novel approach, by providing single molecule images of miRNAs in cellulo can be used as a powerful supplementary tool in the analysis of miRNA function and behaviour and has far reaching potential in defining metastasis-critical subpopulations within a given heterogeneous cancer cell population.