Details about molecular membrane dynamics in living cells, such as lipid-protein interactions, are often hidden from the observer because of the limited spatial resolution of conventional far-field ...optical microscopy. The superior spatial resolution of stimulated emission depletion (STED) nanoscopy can provide new insights into this process. The application of fluorescence correlation spectroscopy (FCS) in focal spots continuously tuned down to 30 nm in diameter distinguishes between free and anomalous molecular diffusion due to, for example, transient binding of lipids to other membrane constituents, such as lipids and proteins. We compared STED-FCS data recorded on various fluorescent lipid analogs in the plasma membrane of living mammalian cells. Our results demonstrate details about the observed transient formation of molecular complexes. The diffusion characteristics of phosphoglycerolipids without hydroxyl-containing headgroups revealed weak interactions. The strongest interactions were observed with sphingolipid analogs, which showed cholesterol-assisted and cytoskeleton-dependent binding. The hydroxyl-containing headgroup of gangliosides, galactosylceramide, and phosphoinositol assisted binding, but in a much less cholesterol- and cytoskeleton-dependent manner. The observed anomalous diffusion indicates lipid-specific transient hydrogen bonding to other membrane molecules, such as proteins, and points to a distinct connectivity of the various lipids to other membrane constituents. This strong interaction is different from that responsible for forming cholesterol-dependent, liquid-ordered domains in model membranes.
The advent of supercontinuum laser sources has enabled the implementation of compact and tunable stimulated emission depletion fluorescence microscopes for imaging far below the diffraction barrier. ...Here we report on an enhanced version of this approach displaying an all-physics based resolution down to (19 ± 3) nm in the focal plane. Alternatively, this single objective lens system can be configured for 3D imaging with resolution down to 45 x 45 x 108 nm in a cell. The obtained results can be further improved by mathematical restoration algorithms. The far-field optical nanoscale resolution is attained in a variety of biological samples featuring strong variations in the local density of features.
Exciting developments: Switching individual photochromic and fluorescent rhodamine amides enables 3D far‐field optical microscopy with nanoscale resolution, excellent signal‐to‐noise ratio, and fast ...acquisition times. The rhodamine amides can be switched on using two photons, which enables 3D detailed imaging of thick and densely stained samples (such as 5‐μm silica beads (see image) and living cells) to be constructed.
A fundamental limit to existing optical techniques for measurement and manipulation of spin degrees of freedom is set by diffraction, which does not allow spins separated by less than about a quarter ...of a micrometre to be resolved using conventional far-field optics. Here, we report an efficient far-field optical technique that overcomes the limiting role of diffraction, allowing individual electronic spins to be detected, imaged and manipulated coherently with nanoscale resolution. The technique involves selective flipping of the orientation of individual spins, associated with nitrogen-vacancy centres in room-temperature diamond, using a focused beam of light with intensity vanishing at a controllable location, which enables simultaneous single-spin imaging and magnetometry at the nanoscale with considerably less power than conventional techniques. Furthermore, by inhibiting spin transitions away from the laser intensity null, selective coherent rotation of individual spins is realized. This technique can be extended to subnanometre dimensions, thus enabling applications in diverse areas ranging from quantum information science to bioimaging. PUBLICATION ABSTRACT
Crack nucleation in negative geometries Weißgraeber, P.; Hell, S.; Becker, W.
Engineering fracture mechanics,
December 2016, 2016-12-00, 20161201, Letnik:
168
Journal Article
Recenzirano
•We analyse structural situations with locally decreasing or increasing energy release rates.•Finite Fracture Mechanics is used to study crack nucleation in three different structural situations.•The ...effects on the nucleation of finite cracks are discussed.•Different stability behaviour of the nucleated cracks is observed and discussed in a general manner.•The results could be used for further investigation of the concept of finite crack advance.
The failure behaviour of structural situations depends on the shape of the energy release rate function of possible crack configurations. Depending on the structural situation, the energy release rate function can decrease (negative geometry) or increase (positive geometry) with increasing crack size. In this work, three structural situations exhibiting locally positive and locally negative behaviour are analysed using a coupled stress and energy criterion in the framework of Finite Fracture Mechanics. The nucleation and stability of finite cracks is discussed in detail. Exact and approximate solutions for the stress fields and the stress intensity factors are employed or newly derived to allow for a closed-form solution of the failure model. For the considered configurations, it is identified whether the nucleated cracks are stable or unstable.
Toward fluorescence nanoscopy Hell, Stefan W
Nature biotechnology,
11/2003, Letnik:
21, Številka:
11
Journal Article
Recenzirano
Odprti dostop
For more than a century, the resolution of focusing light microscopy has been limited by diffraction to 180 nm in the focal plane and to 500 nm along the optic axis. Recently, microscopes have been ...reported that provide three- to sevenfold improved axial resolution in live cells. Moreover, a family of concepts has emerged that overcomes the diffraction barrier altogether. Its first exponent, stimulated emission depletion microscopy, has so far displayed a resolution down to 28 nm. Relying on saturated optical transitions, these concepts are limited only by the attainable saturation level. As strong saturation should be feasible at low light intensities, nanoscale imaging with focused light may be closer than ever.
We carried out a prospective clinical study to evaluate the impact of the Recurrence Score (RS) on treatment decisions in early breast cancer (EBC).
A total of 379 eligible women with estrogen ...receptor positive (ER+), HER2-negative EBC and 0–3 positive lymph nodes were enrolled. Treatment recommendations, patients' decisional conflict, physicians' confidence before and after knowledge of the RS and actual treatment data were recorded.
Of the 366 assessable patients 244 were node negative (N0) and 122 node positive (N+). Treatment recommendations changed in 33% of all patients (N0 30%, N+ 39%). In 38% of all patients (N0 39%, N+ 37%) with an initial recommendation for chemoendocrine therapy, the post-RS recommendation changed to endocrine therapy, in 25% (N0 22%, N+ 39%) with an initial recommendation for endocrine therapy only to combined chemoendocrine therapy, respectively. A patients' decisional conflict score improved by 6% (P = 0.028) and physicians' confidence increased in 45% (P < 0.001) of all cases. Overall, 33% (N0 29%, N+ 38%) of fewer patients actually received chemotherapy as compared with patients recommended chemotherapy pre-test. Using the test was cost-saving versus current clinical practice.
RS-guided chemotherapy decision-making resulted in a substantial modification of adjuvant chemotherapy usage in node-negative and node-positive ER+ EBC.
Abstract Acetylcholine receptor (AChR) supramolecular aggregates that have hitherto only been accessible to examination by electron microscopy were imaged with stimulated emission depletion (STED) ...fluorescence microscopy, providing resolution beyond limits of diffraction of classical wide-field or confocal microscopes. We examined a Chinese hamster ovary cell liner CHO-K1/A5, that stably expresses adult murine AChR. Whereas confocal microscopy displays AChR clusters as diffraction-limited dots of ∼200 nm diameter, STED microscopy yields nanoclusters with a peak size distribution of ∼55 nm. Utilizing this resolution, we show that cholesterol depletion by acute (30 min, 37 °C) exposure to methyl-β-cyclodextrin alters the short and long range organization of AChR nanoclusters on the cell surface. In the short range, AChRs form larger nanoclusters, possibly related to the alteration of cholesterol-dependent protein–protein associations. Ripley’s K-test on STED images reveals changes in nanocluster distribution on larger scales (0.5–3.5 μm), which possibly are related to the abolition of cytoskeletal physical barriers preventing the lateral diffusion of AChR nanoclusters.
Effecting a saturable optical molecular transition with a spatial intensity distribution featuring a local minimum allows the fundamental breaking of the diffraction barrier both in microscopy and in ...material structuring. If the transition can be repeatedly reverted, as in switchable fluorescent proteins and photochromic compounds, fluorescence imaging and writing is possible with spatial resolution down to the molecular scale.
Abstract
Treatment with monoclonal antibodies (mabs) has become an established component of oncological
therapy. The monoclonal antibodies available for this purpose are mainly administered
...intravenously in individually adapted doses according to body weight over longer treatment
times. For other chronic diseases such as, for example, diabetes mellitus, the subcutaneous
administration of drugs is an established therapy option. For the subcutaneous administration of
larger volumes as needed for mab solutions the extracellular matrix of the subcutaneous tissue
represents a problem. The co-formulation with recombinant human hyaluronidase makes the
relatively pain-free administration of larger fluid volumes and thus the subcutaneous
administration of monoclonal antibodies possible, as illustrated by the development of a
subcutaneous formulation of trastuzumab. This constitutes a less invasive, time-optimised and
flexible form of administration for patients with HER2-positive breast cancer that, with its
fixed dosing possibilities, contributes to therapeutic safety. The example of trastuzumab shows
that the subcutaneous administration of monoclonal antibodies can simplify oncological long-term
therapy not only for the patients but also for the medical personnel.
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