Photocaged Hoechst Enables Subnuclear Visualization and Cell Selective Staining of DNA in vivo.

Selective targeting of DNA by means of fluorescent labeling has become a mainstay in the life sciences. While genetic engineering serves as a powerful technique and allows the visualization of nucleic acid by using DNA-targeting fluorescent fusion proteins in a cell-type- and subcellular-specific manner, it relies on the introduction of foreign genes. On the other hand, DNA-binding small fluorescent molecules can be used without genetic engineering, but they are not spatially restricted. Herein, we report a photocaged version of the DNA dye Hoechst33342 (pcHoechst), which can be uncaged by using UV to blue light for the selective staining of chromosomal DNA in subnuclear regions of live cells. Expanding its application to a vertebrate model organism, we demonstrate uncaging in epithelial cells and short-term cell tracking in vivo in zebrafish. We envision pcHoechst as a valuable tool for targeting and interrogating DNA with precise spatiotemporal resolution in living cells and wild-type organisms.

Applicability of solid-phase cytometry and epifluorescence microscopy for rapid assessment of the microbiological quality of dialysis water.

BACKGROUND: Currently, the gold standard to assess the microbiological quality of dialysis water is the determination of heterotrophic plate counts (HPC). The long waiting time of the HPC method and the fact that most bacteria are not culturable on agar plates provokes the search for rapid alternative methods for monitoring the microbiological quality of dialysis water. METHODS: We tested the applicability of total viable counts (TVC) and total direct counts (TDC), determined via solid-phase cytometry and epifluorescence microscopy (EFM), in comparison to the standard HPC determination method in 113 samples from 13 dialysis water treatment units (59 drinking water and 54 dialysis water samples). Additionally, for a set of dialysis water samples (n = 22) endotoxin concentrations were also determined. RESULTS: TVC showed high correlation with HPC and results were of comparable magnitude for most investigated dialysis water samples [median: 3 cells/colony forming units (CFU) 100 mL(-1)]. However, in one dialysis water sample, HPC values (5800 CFU 100 mL(-1)) were >35-fold lower than TVC values (2.05 × 10(5) cells 100 mL(-1)) indicating severe limits of the HPC method to assess the microbiological quality of dialysis water. For drinking water, TVC (median: 4.8 × 10(4) cells 100 mL(-1)) was on average one order of magnitude higher than HPC (median: 2.5 × 10(3) cells 100 mL(-1)). TDCs (median dialysis water: 1.1 × 10(4) cells 100 mL(-1) and median drinking water: 4.9 × 10(6) cells 100 mL(-1)) were always several orders of magnitude higher than HPC or TVC. CONCLUSIONS: We propose that the TVC/solid-phase cytometry approach is a reliable and rapid alternative to the culture-dependent approach for assessment of the microbiological quality of dialysis water, especially when fast results are needed. TDC determined via EFM lacks sensitivity and reliability for assessing microbial concentrations in low-cell dialysis water samples since the limits of detection and quantification are high.

Molecular gradients: an efficient approach for optimizing the surface properties of biomaterials and biochips.

A variety of molecular gradients of alkanethiols with the structure HS-(CH(2))(m)-X (m = 15; X = COOH, CH(2)NH(2), or CH(3)) and oligo(ethylene glycol)-terminated alkanethiols with the structures HS-(CH(2))(15)-CO-NH-Eg(n) (n = 2, 4, or 6), HS-(CH(2))(15)-CO-NH-Eg(2)-(CH(2))(2)-NH-CO-(CH(2))(4)-biotin, and HS-(CH(2))(15)-CO-NH-Eg(6)-CH(2)-COOH were prepared on polycrystalline gold films. These gradients were designed to serve as model surfaces for fundamental studies of protein adsorption and immobilization phenomena. Ellipsometry, infrared spectroscopy, and X-ray photoelectron spectroscopy, operating in scanning mode, were used to monitor the layer composition, gradient profiles, tail group conformation, and overall structural quality of the gradient assemblies. The gradient profiles were found to be 4-10 mm wide, and they increased in width with increasing difference in molecular complexity between the thiols used to form the gradient. The oligo(ethylene glycol) thiols are particularly interesting because they can be used to prepare so-called conformational gradients, that is, gradients that display a variation in oligo(ethylene glycol) chain conformation from all trans on the extreme Eg(2,4) sides, via an amorphous-like phase in the mixing regimes, to helical at the extreme Eg(6) sides. We demonstrate herein a series of experiments where the above gradients are used to evaluate nonspecific binding of the plasma protein fibrinogen, and in agreement with previous studies, the highest amounts of nonspecifically bound fibrinogen were observed on all-trans monolayers, that is, on the extreme Eg(2,4) sides. Moreover, gradients between Eg(2) and a biotinylated analogue have been prepared to optimize the conditions for the immobilization of streptavidin. Ellipsometry and infrared spectroscopy reveal high levels of immobilization over a fairly broad range of compositions in the gradient regime, with a maximum between 50 and 60% of the biotinylated analogue in the monolayer. A pI gradient composed of (NH(3)(+)/COO(-))-terminated thiols was also prepared and evaluated with respect to its ability to separate differently charged proteins, pepsin, and lysozyme, on a solid surface.