Super-resolution re-scan second harmonic generation microscopy.

Second harmonic generation microscopy (SHG) is generally acknowledged as a powerful tool for the label-free three-dimensional visualization of tissues and advanced materials, with one of its most popular applications being collagen imaging. Despite the great need, progress in super-resolved SHG imaging lags behind the developments reported over the past years in fluorescence-based optical nanoscopy. In this work, we demonstrate super-resolved re-scan SHG, qualitatively and quantitatively showing on collagenous tissues the available resolution advantage over the diffraction limit.

Organization and dynamics of the SpoVAEa protein and its surrounding inner membrane lipids, upon germination of Bacillus subtilis spores.

The SpoVA proteins make up a channel in the inner membrane (IM) of Bacillus subtilis spores. This channel responds to signals from activated germinant receptors (GRs), and allows release of Ca-DPA from the spore core during germination. In the current work, we studied the location and dynamics of SpoVAEa in dormant spores.

Depletion of the cellular cholesterol content reduces the dynamics of desmosomal cadherins and interferes with desmosomal strength.

Desmosomal cadherins, desmocollins, and desmogleins are cholesterol-dependent entities responsible for the stable adhesion of desmosomes in epithelial cells. Here, we investigated the influence of cellular cholesterol depletion on the dynamic properties of the desmosomal cadherin desmocollin, particularly the lateral mobility and distribution of desmocollin 2 (Dsc2-YFP) in the plasma membrane, and how these properties influence the adhesion strength of desmosomes. Depletion of cellular cholesterol decreased the lateral mobility of Dsc2-YFP and caused dispersion of Dsc2-YFP in the plasma membrane of epithelial MDCK cells.

Visualization of Germinosomes and the Inner Membrane in Bacillus subtilis Spores.

The small size of spores and the relatively low abundance of germination proteins, cause difficulties in their microscopic analyses using epifluorescence microscopy. Super-resolution three-dimensional Structured Illumination Microscopy (3D-SIM) is a promising tool to overcome this hurdle and reveal the molecular details of the process of germination of Bacillus subtilis (B. subtilis) spores.

Fluorescence-detected linear dichroism imaging in a re-scan confocal microscope equipped with differential polarization attachment.

Confocal laser scanning microscopy is probably the most widely used and one of the most powerful techniques in basic biology, medicine and material sciences that is employed to elucidate the architecture of complex cellular structures and molecular macro-assemblies. It has recently been shown that the information content, signal-to-noise ratio and resolution of such microscopes (LSMs) can be improved significantly by adding different attachments or modifying their design, while retaining their user-friendly features and relatively moderate costs. Differential polarization (DP) attachments, using high-frequency modulation/demodulation circuits, have made LSMs capable of high-precision 2D and 3D mapping of the anisotropy of microscopic samples-without interfering with their 'conventional' fluorescence or transmission imaging (Steinbach et al.

Helical organization of microtubules occurs in a minority of tunneling membrane nanotubes in normal and cancer urothelial cells.

Tunneling membrane nanotubes (TnTs) are membrane protrusions connecting nearby or distant cells in vitro and in vivo. Functions of TnTs in cellular processes are various and rely on TnT structure, which also depends on cytoskeletal composition. In the present study, we focused on the organization of microtubules (MTs) and intermediate filaments (IFs) in TnTs of urothelial cells.

Re-scan confocal microscopy (RCM) improves the resolution of confocal microscopy and increases the sensitivity.

Re-scan confocal microscopy (RCM) is a new super-resolution technique based on a standard confocal microscope extended with a re-scan unit in the detection path that projects the emitted light onto a sensitive camera. In this paper the fundamental properties of RCM, lateral resolution, axial resolution and signal-to-noise ratio, are characterized and compared with properties of standard confocal microscopy. The results show that the lateral resolution of RCM is ~170 nm compared to ~240 nm of confocal microscopy for 488 nm excitation and 1.

Intracellular pH Response to Weak Acid Stress in Individual Vegetative Bacillus subtilis Cells.

Unlabelled: Intracellular pH (pH) critically affects bacterial cell physiology. Hence, a variety of food preservation strategies are aimed at perturbing pH homeostasis. Unfortunately, accurate pH quantification with existing methods is suboptimal, since measurements are averages across populations of cells, not taking into account interindividual heterogeneity.

Quantifying the effect of sorbic acid, heat and combination of both on germination and outgrowth of Bacillus subtilis spores at single cell resolution.

Bacillus subtilis spores are a problem for the food industry as they are able to survive preservation processes. The spores often reside in food products, where their inherent protection against various stress treatments causes food spoilage. Sorbic acid is widely used as a weak acid preservative in the food industry.

The molecular architecture of hemidesmosomes, as revealed with super-resolution microscopy.

Hemidesmosomes have been extensively studied with immunofluorescence microscopy, but owing to its limited resolution, the precise organization of hemidesmosomes remains poorly understood. We studied hemidesmosome organization in cultured keratinocytes with two- and three-color super-resolution microscopy. We observed that, in the cell periphery, nascent hemidesmosomes are associated with individual keratin filaments and that β4 integrin (also known as ITGB4) is distributed along, rather than under, keratin filaments.

Co-Orientation: Quantifying Simultaneous Co-Localization and Orientational Alignment of Filaments in Light Microscopy.

Co-localization analysis is a widely used tool to seek evidence for functional interactions between molecules in different color channels in microscopic images. Here we extend the basic co-localization analysis by including the orientations of the structures on which the molecules reside. We refer to the combination of co-localization of molecules and orientational alignment of the structures on which they reside as co-orientation.

Caenorhabditis elegans as a Model System for Studying Drug Induced Mitochondrial Toxicity.

Today HIV-1 infection is recognized as a chronic disease with obligatory lifelong treatment to keep viral titers below detectable levels. The continuous intake of antiretroviral drugs however, leads to severe and even life-threatening side effects, supposedly by the deleterious impact of nucleoside-analogue type compounds on the functioning of the mitochondrial DNA polymerase. For detailed investigation of the yet partially understood underlying mechanisms, the availability of a versatile model system is crucial.

In vivo visualization and quantification of mitochondrial morphology in C. elegans.

Caenorhabditis elegans is a highly malleable model system, intensively used for functional, genetic, cytometric, and integrative studies. Due to its simplicity and large muscle cell number, C. elegans has frequently been used to study mitochondrial deficiencies caused by disease or drug toxicity.

Quantitative analysis of the effect of specific tea compounds on germination and outgrowth of Bacillus subtilis spores at single cell resolution.

Tea is one of the most widely consumed beverages in the world and known for its antimicrobial activity against many microorganisms. Preliminary studies have shown that tea polyphenols can inhibit the growth of a wide range of Gram-positive bacteria. However, the effect of these compounds on germination and outgrowth of bacterial spores is unclear.

Dynamics of the establishment of multinucleate compartments in Fusarium oxysporum.

Nuclear dynamics can vary widely between fungal species and between stages of development of fungal colonies. Here we compared nuclear dynamics and mitotic patterns between germlings and mature hyphae in Fusarium oxysporum. Using fluorescently labeled nuclei and live-cell imaging, we show that F.

Depletion of the chromatin looping proteins CTCF and cohesin causes chromatin compaction: insight into chromatin folding by polymer modelling.

Folding of the chromosomal fibre in interphase nuclei is an important element in the regulation of gene expression. For instance, physical contacts between promoters and enhancers are a key element in cell-type-specific transcription. We know remarkably little about the principles that control chromosome folding.

Imaging dendritic spines of rat primary hippocampal neurons using structured illumination microscopy.

Dendritic spines are protrusions emerging from the dendrite of a neuron and represent the primary postsynaptic targets of excitatory inputs in the brain. Technological advances have identified these structures as key elements in neuron connectivity and synaptic plasticity. The quantitative analysis of spine morphology using light microscopy remains an essential problem due to technical limitations associated with light's intrinsic refraction limit.

Robustness of DNA repair through collective rate control.

DNA repair and other chromatin-associated processes are carried out by enzymatic macromolecular complexes that assemble at specific sites on the chromatin fiber. How the rate of these molecular machineries is regulated by their constituent parts is poorly understood. Here we quantify nucleotide-excision DNA repair in mammalian cells and find that, despite the pathways' molecular complexity, repair effectively obeys slow first-order kinetics.

Filamin A promotes dynamin-dependent internalization of hyperpolarization-activated cyclic nucleotide-gated type 1 (HCN1) channels and restricts Ih in hippocampal neurons.

The actin-binding protein filamin A (FLNa) regulates neuronal migration during development, yet its roles in the mature brain remain largely obscure. Here, we probed the effects of FLNa on the regulation of ion channels that influence neuronal properties. We focused on the HCN1 channels that conduct Ih, a hyperpolarization-activated current crucial for shaping intrinsic neuronal properties.

Re-scan confocal microscopy: scanning twice for better resolution.

We present a new super-resolution technique, Re-scan Confocal Microscopy (RCM), based on standard confocal microscopy extended with an optical (re-scanning) unit that projects the image directly on a CCD-camera. This new microscope has improved lateral resolution and strongly improved sensitivity while maintaining the sectioning capability of a standard confocal microscope. This simple technology is typically useful for biological applications where the combination high-resolution and high-sensitivity is required.

Live cell imaging of germination and outgrowth of individual bacillus subtilis spores; the effect of heat stress quantitatively analyzed with SporeTracker.

Spore-forming bacteria are a special problem for the food industry as some of them are able to survive preservation processes. Bacillus spp. spores can remain in a dormant, stress resistant state for a long period of time.

Distinct regional and subcellular localization of the actin-binding protein filamin A in the mature rat brain.

Filamin A (FLNa) is an actin-binding protein that regulates cell motility, adhesion, and elasticity by cross-linking filamentous actin. Additional roles of FLNa include regulation of protein trafficking and surface expression. Although the functions of FLNa during brain development are well studied, little is known on its expression, distribution, and function in the adult brain.

Repetitive disruptions of the nuclear envelope invoke temporary loss of cellular compartmentalization in laminopathies.

The nuclear lamina provides structural support to the nucleus and has a central role in nuclear organization and gene regulation. Defects in its constituents, the lamins, lead to a class of genetic diseases collectively referred to as laminopathies. Using live cell imaging, we observed the occurrence of intermittent, non-lethal ruptures of the nuclear envelope in dermal fibroblast cultures of patients with different mutations of lamin A/C.

Real-time visualization of heterotrimeric G protein Gq activation in living cells.

Background: Gq is a heterotrimeric G protein that plays an important role in numerous physiological processes. To delineate the molecular mechanisms and kinetics of signalling through this protein, its activation should be measurable in single living cells. Recently, fluorescence resonance energy transfer (FRET) sensors have been developed for this purpose.