Preventing cation intermixing enables 50% quantum yield in sub-15 nm short-wave infrared-emitting rare-earth based core-shell nanocrystals

Short-wave infrared (SWIR) fluorescence could become the new gold standard in optical imaging for biomedical applications due to important advantages such as lack of autofluorescence, weak photon absorption by blood and tissues, and reduced photon scattering coefficient. Therefore, contrary to the visible and NIR regions, tissues become translucent in the SWIR region. Nevertheless, the lack of bright and biocompatible probes is a key challenge that must be overcome to unlock the full potential of SWIR fluorescence. Although rare-earth-based core-shell nanocrystals appeared as promising SWIR probes, they suffer from limited photoluminescence quantum yield (PLQY). The lack of control over the atomic scale organization of such complex materials is one of the main barriers limiting their optical performance. Here, the growth of either homogeneous (α-NaYF$_4$) or heterogeneous (CaF$_2$) shell domains on optically-active α-NaYF$_4$:Yb:Er (with and without Ce$^{3+}$ co-doping) core nanocrystals is reported. The atomic scale organization can be controlled by preventing cation intermixing only in heterogeneous core-shell nanocrystals with a dramatic impact on the PLQY. The latter reached 50% at 60 mW/cm$^2$; one of the highest reported PLQY values for sub-15 nm nanocrystals. The most efficient nanocrystals were utilized for in vivo imaging above 1450 nm

Catalytic Conversion of Lipophilic Substrates by Phase constrained Enzymes in the Aqueous or in the Membrane Phase

Both soluble and membrane-bound enzymes can catalyze the conversion of lipophilic substrates. The precise substrate access path, with regard to phase, has however, until now relied on conjecture from enzyme structural data only (certainly giving credible and valuable hypotheses). Alternative methods have been missing. To obtain the first experimental evidence directly determining the access paths (of lipophilic substrates) to phase constrained enzymes we here describe the application of a BODIPY-derived substrate (PS1). Using this tool, which is not accessible to cytosolic enzymes in the presence of detergent and, by contrast, not accessible to membrane embedded enzymes in the absence of detergent, we demonstrate that cytosolic and microsomal glutathione transferases (GSTs), both catalyzing the activation of PS1, do so only within their respective phases. This approach can serve as a guideline to experimentally validate substrate access paths, a fundamental property of phase restricted enzymes. Examples of other enzyme classes with members in both phases are xenobiotic-metabolizing sulphotransferases/UDP-glucuronosyl transferases or epoxide hydrolases. Since specific GSTs have been suggested to contribute to tumor drug resistance, PS1 can also be utilized as a tool to discriminate between phase constrained members of these enzymes by analyzing samples in the absence and presence of Triton X-100

Photoswitching Mechanism of Cyanine Dyes

Photoswitchable fluorescent probes have been used in recent years to enable super-resolution fluorescence microscopy by single-molecule imaging.1-6 Among these probes are red carbocyanine dyes, which can be reversibly photoconverted between a fluorescent state and a dark state for hundreds of cycles, yielding several thousand detected photons per switching cycle, before permanent photobleaching occurs.7,8 While these properties make them excel-lent probes for super-resolution imaging, the mechanism by which cyanine dyes are photoconverted has yet to be determined. Such an understanding could prove useful for creating new photoswit-chable probes with improved properties. The photoconversion of red cyanine dyes into their dark states occurs upon illumination by red light and is facilitated by a primary thiol in solution,7,9 whereas agents with a secondary thiol do not support photoswitching. These observations suggest that the reactiv

Fluorescent Labeling of SNAP-Tagged Proteins in Cells

One of the most prominent self-labeling tags is SNAP-tag. It is an in vitro evolution product of the human DNA repair protein O6 -alkylguanine-DNA alkyltransferase (hAGT) that reacts specifically with benzylguanine (BG) and benzylchloropyrimidine (CP) derivatives, leading to covalent labeling of SNAP-tag with a synthetic probe (Gronemeyer et al., Protein Eng Des Sel 19:309–316, 2006; Curr Opin Biotechnol 16:453–458, 2005; Keppler et al., Nat Biotechnol 21:86–89, 2003; Proc Natl Acad Sci U S A 101:9955– 9959, 2004). SNAP-tag is well suited for the analysis and quantification of fused target protein using fluorescence microscopy techniques. It provides a simple, robust, and versatile approach to the imaging of fusion proteins under a wide range of experimental conditions. © Springer Science+Business Media New York 2015

Coevolution of nutrigenomics and society: ethical considerations

To optimize the coevolution of nutrigenomics and society (ie, the reciprocal stimulation of both developments), I analyzed chances for a fruitful match between normative concepts and strategies of both developments. Nutrigenomics embodies =3 normative concepts. First, food is exclusively interpreted in terms of disease prevention. Second, striving for health is interpreted as the quantification of risks and prevention of diseases through positive food-gene interactions. The third normative idea is that disease prevention by the minimization of risks is an individual's task. My thesis was that these concepts of nutrigenomics would not easily match with concepts of food and health of various food styles in Western societies, which, for instance, parents in the case of metabolic programming endorse and with a philosophical view of the relation between food, health, and the meaning of life. Next, I reflected on the nonsynchronized coevolution of nutrigenomics and society because of this mismatch and introduced the concept of the fair representation of food styles in nutrigenomic developments. To synchronize and optimize the coevolution of nutrigenomics and society, I propose that the research policy of nutrigenomics should change to a research partnership with society on the basis of fair representation

Resolft nanoscopy in living cells at high speed

Among various superresolution methods, RESOLFT nanoscopy has advantages in compatibility with live cell imaging via utilization of genetically encoded reversely-switchable fluorescent proteins (rsFPs). Recent developments in rsFPs allow nanoscopy in living cells at high speed. ©Optics in the Life Sciences Congress Technical Digest

Strategies to improve photostabilities in ultrasensitive fluorescence spectroscopy.

Given the particular importance of dye photostability for single-molecule and fluorescence fluctuation spectroscopy investigations, refined strategies were explored for how to chemically retard dye photobleaching. These strategies will be useful for fluorescence correlation spectroscopy (FCS), fluorescence-based confocal single-molecule detection (SMD) and related techniques. In particular, the effects on the addition of two main categories of antifading compounds, antioxidants (n-propyl gallate, nPG, ascorbic acid, AA) and triplet state quenchers (mercaptoethylamine, MEA, cyclo-octatetraene, COT), were investigated, and the relevant rate parameters involved were determined for the dye Rhodamine 6G. Addition of each of the compound categories resulted in significant improvements in the fluorescence brightness of the monitored fluorescent molecules in FCS measurements. For antioxidants, we identify the balance between reduction of photoionized fluorophores on the one hand and that of intact fluorophores on the other as an important guideline for what concentrations to be added for optimal fluorescence generation in FCS and SMD experiments. For nPG/AA, this optimal concentration was found to be in the lower micromolar range, which is considerably less than what has previously been suggested. Also, for MEA, which is a compound known as a triplet state quencher, it is eventually its antioxidative properties and the balance between reduction of fluorophore cation radicals and that of intact fluorophores that defines the optimal added concentration. Interestingly, in this optimal concentration range the triplet state quenching is still far from sufficient to fully minimize the triplet populations. We identify photoionization as the main mechanism of photobleaching within typical transit times of fluorescent molecules through the detection volume in a confocal FCS or SMD instrument (<1-20 ms), and demonstrate its generation via both one- and multistep excitation processes. Apart from reflecting a major pathway for photobleaching, our results also suggest the exploitation of the photoinduced ionization and the subsequent reduction by antioxidants for biomolecular monitoring purposes and as a possible switching mechanism with applications in high-resolution microscopy