Alexander Benke

Cell Morphology and Intracellular Ionic Homeostasis explored with a Multimodal Approach combining Epifluorescence and Digital Holographic Microscopy

The authors have developed a live-cell multimodality microscope combining epifluorescence with digital holographic microscopy; it has been implemented with a decoupling procedure allowing to separately measure from the quantitative phase important cell parameters including absolute volume, shape and integral intracellular refractive index. In combination with the numerous different specific fluorescent cellular probes, this multimodality microscopy can address important issues in cell biology. This is demonstrated by the study of intracellular calcium homeostasis associated with the change in cell volume, which play a critical role in the excitotoxicity-induced neuronal death.

Live-Cell dSTORM of Cellular DNA Based on Direct DNA Labeling

We have implemented the super-resolution method of direct stochastic optical reconstruction microscopy (dSTORM) to image nuclear and mitochondrial DNA in living cells. We also demonstrate time-lapse imaging, all using a dye that associates directly with cellular DNA: the commercially available dye Picogreen

Nanoscale spatial organization of the HoxD gene cluster in distinct transcriptional states.

Significance Ultrastructural chromatin dynamics may play a key role in regulating transcriptional activation. Here we have used super-resolution microscopy to study the folding mechanics of the HoxD cluster, as assayed by following the elongation of chromatin in single cells with different status of Hox gene activation. We observed that the spatial separation of Hoxd genes is strongest in those tissues where they are highly expressed. We also document that the opening of chromatin precedes transcription and that the strongest elongations are observed at the location of the boundary between two major topologically associating domains (TADs). These results shed light on how spatial compartmentalization is achieved, likely to accompany efficient chromatin reorganization upon activation of transcriptional switches. Chromatin condensation plays an important role in the regulation of gene expression. Recently, it was shown that the transcriptional activation of Hoxd genes during vertebrate digit development involves modifications in 3D interactions within and around the HoxD gene cluster. This reorganization follows a global transition from one set of regulatory contacts to another, between two topologically associating domains (TADs) located on either side of the HoxD locus. Here, we use 3D DNA FISH to assess the spatial organization of chromatin at and around the HoxD gene cluster and report that although the two TADs are tightly associated, they appear as spatially distinct units. We measured the relative position of genes within the cluster and found that they segregate over long distances, suggesting that a physical elongation of the HoxD cluster can occur. We analyzed this possibility by super-resolution imaging (STORM) and found that tissues with distinct transcriptional activity exhibit differing degrees of elongation. We also observed that the morphological change of the HoxD cluster in developing digits is associated with its position at the boundary between the two TADs. Such variations in the fine-scale architecture of the gene cluster suggest causal links among its spatial configuration, transcriptional activation, and the flanking chromatin context.

Multicolor single molecule tracking of stochastically active synthetic dyes.

Single particle tracking can reveal dynamic information at the scale of single molecules in living cells but thus far has been limited either in the range of potential protein targets or in the quality and number of tracks attainable. We demonstrate a new approach to single molecule tracking by using the blinking properties of synthetic dyes targeted to proteins of interest with genetically encoded tags to generate high-density tracks while maintaining flexibility in protein labeling. We track membrane proteins using different combinations of dyes and show that the concept can be extended to three-color imaging. Moreover, we show that this technique is not limited to the membrane by performing live tracking of proteins in intracellular compartments.

In Situ Characterization of Bak Clusters Responsible for Cell Death Using Single Molecule Localization Microscopy

Apoptosis plays a pivotal role in development and tissue homeostasis in multicellular organisms. Clustering of Bak proteins on the mitochondrial outer membrane is responsible for the induction of apoptosis by evoking a release of pro-apoptotic proteins from mitochondria into cytosol. However, how the protein cluster permeabilizes the mitochondrial membrane remains unclear because elucidation of the cluster characteristics such as size and protein density has been hampered by the diffraction-limited resolution of light microscopy. Here, we describe an approach to quantitatively characterize Bak clusters in situ based on single molecule localization. We showed that Bak proteins form densely packed clusters at the nanoscale on mitochondria during apoptosis. Quantitative analysis based on the localization of each Bak protein revealed that the density of Bak protein is uniform among clusters although the cluster size is highly heterogeneous. Our approach provides unprecedented information on the size and protein density of Bak clusters possibly critical for the permeabilization and is applicable for the analysis of different cluster formations.

Visualizing the HoxD Gene Cluster at the Nanoscale Level

Transcription of HoxD cluster genes in limbs is coordinated by two topologically associating domains (TADs), neighboring the cluster and containing various enhancers. Here, we use a combination of microscopy approaches and chromosome conformation capture to assess the structural changes occurring in this global architecture in various functional states. We observed that despite their spatial juxtaposition, the TADs are consistently kept as distinct three-dimensional units. Hox genes located at their boundary can show significant spatial segregation over long distances, suggesting that physical elongation of the HoxD cluster occurs. The use of superresolution imaging (STORM [stochastic optical reconstruction microscopy]) revealed that the gene cluster can be in an either compact or elongated shape. The latter configuration is observed in transcriptionally active tissue and in embryonic stem cells, consistent with chromosome conformation capture results. Such morphological changes at HoxD in developing digits seem to be associated with its position at the boundary between two TADs and support the idea that chromatin dynamics is important in the establishment of transcriptional activity.

Reduced Dyes Enhance Single-Molecule Localization Density for Live Superresolution Imaging

Cell-permeable rhodamine dyes are reductively quenched by NaBH4 into a non-fluorescent leuco-rhodamine form. Quenching is reversible, and their fluorescence is recovered when the dyes are oxidized. In living cells, oxidation occurs spontaneously, and can result in up to ten-fold higher densities of single molecule localizations, and more photons per localization as compared with unmodified dyes. These two parameters directly impact the achievable resolution, and we see a significant improvement in the quality of live-cell point-localization super-resolution images taken with reduced dyes. These improvements carry over to increase the density of trajectories for single-molecule tracking experiments.

Study of Intracellular Ion Dynamics with a Multimodality Approach Combining Epifluorescence and Digital Holographic Microscopy

We present combined measurements of quantitative phase through digital holographic microscopy (DHM) and epifluorescence for cells dynamics study. We concentrate our investigation on intracellular ion concentration monitoring with both techniques for comparison.