Initial photophysical characterization of the proteorhodopsin optical proton sensor (PROPS)

Fluorescence is not frequently used as a tool for investigating the photocycles of rhodopsins, largely because of the low quantum yield of the retinal chromophore. However, a new class of genetically encoded voltage sensors is based upon rhodopsins and their fluorescence. The first such sensor reported in the literature was the proteorhodopsin optical proton sensor (PROPS), which is capable of indicating membrane voltage changes in bacteria by means of changes in fluorescence. However, the properties of this fluorescence, such as its lifetime decay components and its origin in the protein photocycle, remain unknown. This paper reports steady-state and nanosecond time-resolved emission of this protein expressed in two strains of Escherichia coli, before and after membrane depolarization. The voltage-dependence of a particularly long lifetime component is established. Additional work to improve quantum yields and improve the general utility of PROPS is suggested

Differential toxicity of gold-doxorubicin in cancer cells vs. cardiomyocytes as measured by real-time growth assays and fluorescence lifetime imaging microscopy (FLIM)

The kinetics of toxicity of doxorubicin (Dox) and gold nanoparticle-conjugated doxorubicin (Au-Dox) were investigated in cultured B16 melanoma cells and cardiomyocytes using real-time cell-growth imaging. Both bolus exposure and continuous exposure were used. Modeling of the growth curve dynamics suggested patterns of uptake and/or expulsion of the drug that were different for the different cell lines and exposures. Dox alone in B16 cells fit to a model of slow drug buildup, whereas Au-Dox fit to a pattern of initial high drug efficacy followed by a decrease. In cardiomyocytes, the best fit was to a model of increasing drug concentration which then began to decrease, consistent with breakdown of the doxorubicin in solution. Cardiomyocytes were more sensitive than B16 cells to Dox alone (IC_(50) 123 ± 2 nM vs. 270 ± 2 nM with continuous exposure), but were dramatically less sensitive to Au-Dox (IC_(50) 1 ± 0.1 μM vs. 58 ± 5 nM with continuous exposure). Bolus exposure for 40 min led to significant cell death in B16 cells but not in cardiomyocytes. Fluorescence lifetime imaging (FLIM) showed different patterns of uptake of Au-Dox in the two cell types that explained the differential toxicity. While Au-Dox concentrated in the nuclei of B16 cells, it remained endosomal in cardiomyocytes. These results suggest that stable conjugates of nanoparticles to doxorubicin may be useful for treating resistant cancers while sparing healthy tissue

Use of dyes to increase phase contrast for biological holographic microscopy

Holographic microscopy is an emerging biological technique that provides amplitude and quantitative phase imaging, though the contrast provided by many cell types and organelles is low, and until now no dyes were known that increased contrast. Here we show that the metallocorrole Ga(tpfc)(SO_3H)_2, which has a strong Soret band absorption, increases contrast in both amplitude and phase and facilitates tracking of Escherichia coli with minimal toxicity. The change in phase contrast may be calculated from the dye-absorbance spectrum using the Kramers–Kronig relations, and represents a general principle that may be applied to any dye or cell type. This enables the use of holographic microscopy for all applications in which specific labeling is desired

Progress in Development of Improved Ion-Channel Biosensors

Further improvements have recently been made in the development of the devices described in Improved Ion-Channel Biosensors (NPO-30710), NASA Tech Briefs, Vol. 28, No. 10 (October 2004), page 30. As discussed in more detail in that article, these sensors offer advantages of greater stability, greater lifetime, and individual electrical addressability, relative to prior ion-channel biosensors. In order to give meaning to a brief description of the recent improvements, it is necessary to recapitulate a substantial portion of the text of the cited previous article. The figure depicts one sensor that incorporates the recent improvements, and can be helpful in understanding the recapitulated text, which follows: These sensors are microfabricated from silicon and other materials compatible with silicon. Typically, the sensors are fabricated in arrays in silicon wafers on glass plates. Each sensor in the array can be individually electrically addressed, without interference with its neighbors. Each sensor includes a well covered by a thin layer of silicon nitride, in which is made a pinhole for the formation of a lipid bilayer membrane. In one stage of fabrication, the lower half of the well is filled with agarose, which is allowed to harden. Then the upper half of the well is filled with a liquid electrolyte (which thereafter remains liquid) and a lipid bilayer is painted over the pinhole. The liquid contains a protein that forms an ion channel on top of the hardened agarose. The combination of enclosure in the well and support by the hardened agarose provides the stability needed to keep the membrane functional for times as long as days or even weeks. An electrode above the well, another electrode below the well, and all the materials between the electrodes together constitute a capacitor. What is measured is the capacitive transient current in response to an applied voltage pulse. One notable feature of this sensor, in comparison with prior such sensors, is a relatively thick dielectric layer between the top of the well and the top electrode. This layer greatly reduces the capacitance of an aperture across which the ion channels are formed, thereby increasing the signal-to-noise ratio. The use of a relatively large aperture with agarose support makes it possible to form many ion channels instead of only one, thereby further increasing the signal-to-noise ratio and effectively increasing the size of the available ionic reservoir. The relatively large reservoir makes it possible to measure AC rather than DC. This concludes the recapitulation from the cited previous article

Imaging technologies and strategies for detection of extant extraterrestrial microorganisms

There is no reductionist definition of life, so the way organisms look, behave, and move is the most definitive way to identify extraterrestrial life. Life elsewhere in the Solar System is likely to be microbial, but no microscope capable of imaging prokaryotic life has ever flown on a lander mission to a habitable planet. Nonetheless, high-resolution microscopes have been developed that are appropriate for planetary exploration. Traditional light microscopy, interferometric microscopy, light-field microscopy, scanning probe microscopy, and electron microscopy are all possible techniques for the detection of extant micro-organisms on Mars and the moons of Jupiter and Saturn. This article begins with a general discussion of the challenges involved in searching for prokaryotic life, then reviews instruments that have flown, that have been selected for flight but not flown or not flown yet, and developing techniques of great promise for life detection that have not yet been selected for flight

Fluorescence Intensity and Intermittency as Tools for Following Dopamine Bioconjugate Processing in Living Cells

CdSe/ZnS quantum dots (QDs) conjugated to biomolecules that quench their fluorescence, particularly dopamine, have particular spectral properties that allow determination of the number of conjugates per particle, namely, photoenhancement and photobleaching. In this work, we quantify these properties on a single-particle and ensemble basis in order to evaluate their usefulness as a tool for indicating QD uptake, breakdown, and processing in living cells. This creates a general framework for the use of fluorescence quenching and intermittency to better understand nanoparticle-cell interactions

InP/ZnS as a safer alternative to CdSe/ZnS core/shell quantum dots: in vitro and in vivo toxicity assessment

We show that water soluble InP/ZnS core/shell QDs are a safer alternative to CdSe/ZnS QDs for biological applications, by comparing their toxicity in vitro (cell culture) and in vivo (animal model Drosophila). By choosing QDs with comparable physical and chemical properties, we find that cellular uptake and localization are practically identical for these two nanomaterials. Toxicity of CdSe/ZnS QDs appears to be related to the release of poisonous Cd(2+) ions and indeed we show that there is leaching of Cd(2+) ions from the particle core despite the two-layer ZnS shell. Since an almost identical amount of In(III) ions is observed to leach from the core of InP/ZnS QDs, their very low toxicity as revealed in this study hints at a much lower intrinsic toxicity of indium compared to cadmium

Novel sulfur-oxidizing streamers thriving in a perennial cold saline springs of the Canadian high Arctic

The perennial springs at Gypsum Hill (GH) and Colour Peak (CP), situated at nearly 80\ub0N on Axel Heiberg Island in the Canadian high Arctic, are one of the few known examples of cold springs in thick permafrost on Earth. The springs emanate from deep saline aquifers and discharge cold anoxic brines rich in both sulfide and sulfate. Grey-coloured microbial streamers form during the winter months in snow-covered regions of the GH spring run-off channels (-1.3\ub0C to 6.9\ub0C, ~7.5% NaCl, 0\u201320 p.p.m. dissolved sulfide, 1 p.p.m. dissolved oxygen) but disappear during the Arctic summer. Culture- and molecular-based analyses of the 16S rRNA gene (FISH, DGGE and clone libraries) indicated that the streamers were uniquely dominated by chemolithoautotrophic sulfur-oxidizing Thiomicrospira species. The streamers oxidized both sulfide and thiosulfate and fixed CO2 under in situ conditions and a Thiomicrospira strain isolated from the streamers also actively oxidized sulfide and thiosulfate and fixed CO2 under cold, saline conditions. Overall, the snow-covered spring channels appear to represent a unique polar saline microhabitat that protects and allows Thiomicrospira streamers to form and flourish via chemolithoautrophic, phototrophicindependent metabolism in a high Arctic winter environment characterized by air temperatures commonly below -40\ub0C and with an annual average air temperature of -15\ub0C. These results broaden our knowledge of the physical and chemical boundaries that define life on Earth and have astrobiological implications for the possibility of life existing under similar Martian conditions.NRC publication: Ye