The use of fluorescence lifetime imaging (FLIM) for in situ microbial detection in complex mineral substrates.

The utility of fluorescence lifetime imaging microscopy (FLIM) for identifying bacteria in complex mineral matrices was investigated. Baseline signals from unlabelled Bacillus subtilis and Euglena gracilis, and Bacillus subtilis labelled with SYTO 9 were obtained using two-photon excitation at 730, 750 and 800 nm, identifying characteristic lifetimes of photosynthetic pigments, unpigmented cellular autofluorescence, and SYTO 9. Labelled and unlabelled B. subtilis were seeded onto marble and gypsum samples containing endolithic photosynthetic cyanobacteria and the ability to distinguish cells from mineral autofluorescence and nonspecific dye staining was examined in parallel with ordinary multichannel confocal imaging. It was found that FLIM enabled discrimination of SYTO 9 labelled cells from background, but that the lifetime of SYTO 9 was shorter in cells on minerals than in pure culture under our conditions. Photosynthetic microorganisms were easily observed using both FLIM and confocal. Unlabelled, nonpigmented bacteria showed weak signals that were difficult to distinguish from background when minerals were present, though cellular autofluorescence consistent with NAD(P)H could be seen in pure cultures, and phasor analysis permitted detection on rocks. Gypsum and marble samples showed similar autofluorescence profiles, with little autofluorescence in the yellow-to-red range. Lifetime or time-gated imaging may prove a useful tool for environmental microbiology. LAY DESCRIPTION: The standard method of bacterial enumeration is to label the cells with a fluorescent dye and count them under high-power fluorescence microscopy. However, this can be difficult when the cells are embedded in soil and rock due to fluorescence from the surrounding minerals and dye binding to ambiguous features of the substrate. The use of fluorescence lifetime imaging (FLIM) can disambiguate these signals and allow for improved detection of bacteria in environmental samples.

Detectability of unresolved particles in off-axis digital holographic microscopy.

Off-axis digital holographic microscopy (DHM) provides both amplitude and phase images, and so it may be used for label-free 3D tracking of micro- and nano-sized particles of different compositions, including biological cells, strongly absorbing particles, and strongly scattering particles. Contrast is provided by differences in either the real or imaginary parts of the refractive index (phase contrast and absorption) and/or by scattering. While numerous studies have focused on phase contrast and improving resolution in DHM, particularly axial resolution, absent have been studies quantifying the limits of detection for unresolved particles. This limit has important implications for microbial detection, including in life-detection missions for space flight. Here we examine the limits of detection of nanosized particles as a function of particle optical properties, microscope optics (including camera well depth and substrate), and data processing techniques and find that DHM provides contrast in both amplitude and phase for unresolved spheres, in rough agreement with Mie theory scattering cross-sections. Amplitude reconstructions are more useful than phase for low-index spheres and should not be neglected in DHM analysis.

Using the Gouy phase anomaly to localize and track bacteria in digital holographic microscopy 4D images.

Described over 100 years ago, the Gouy phase anomaly refers to the additional π phase shift that is accumulated as a wave passes through focus. It is potentially useful in analyzing any type of phase-sensitive imaging; in light microscopy, digital holographic microscopy (DHM) provides phase information in the encoded hologram. One limitation of DHM is the weak contrast generated by many biological cells, especially unpigmented bacteria. We demonstrate here that the Gouy phase anomaly may be detected directly in the phase image using the z-derivative of the phase, allowing for precise localization of unlabeled, micrometer-sized bacteria. The use of dyes that increase phase contrast does not improve detectability. This approach is less computationally intensive than other procedures such as deconvolution and is relatively insensitive to reconstruction parameters. The software is implemented in an open-source FIJI plug-in.

Genetically Encoded Phase Contrast Agents for Digital Holographic Microscopy.

Quantitative phase imaging and digital holographic microscopy have shown great promise for visualizing the motion, structure, and physiology of microorganisms and mammalian cells in three dimensions. However, these imaging techniques currently lack molecular contrast agents analogous to the fluorescent dyes and proteins that have revolutionized fluorescence microscopy. Here we introduce the first genetically encodable phase contrast agents based on gas vesicles. The relatively low index of refraction of the air-filled core of gas vesicles results in optical phase advancement relative to aqueous media, making them a "positive" phase contrast agent easily distinguished from organelles, dyes, or microminerals. We demonstrate this capability by identifying and tracking the motion of gas vesicles and gas vesicle-expressing bacteria using digital holographic microscopy, and by imaging the uptake of engineered gas vesicles by mammalian cells. These results give phase imaging a biomolecular contrast agent, expanding the capabilities of this powerful technology for three-dimensional biological imaging.

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)(SO3)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.

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 (IC50 123 ± 2 nM vs. 270 ± 2 nM with continuous exposure), but were dramatically less sensitive to Au-Dox (IC50 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.

Targeting B16 tumors in vivo with peptide-conjugated gold nanoparticles.

This study examines the effects of polyethylene glycol (PEG) and peptide conjugation on the biodistribution of ultrasmall (2.7 nm) gold nanoparticles in mice bearing B16 melanoma allografts. Nanoparticles were delivered intravenously, and biodistribution was measured at specific timepoints by organ digestion and inductively coupled plasma mass spectrometry. All major organs were examined. Two peptides were tested: the cyclic RGD peptide (cRGD, which targets integrins); and a recently described peptide derived from the myxoma virus. We found the greatest specific tumor delivery using the myxoma peptide, with or without PEGylation. Un-PEGylated cRGD performed poorly, but PEGylated RGD showed a significant transient collection in the tumor. Liver and kidney were the primary targets of all constructs. None of the particles were able to cross the blood-brain barrier. Although it was able to deliver Au to B16 cells, the myxoma peptide did not show any cytotoxic activity against these cells, in contrast to previous reports. These results indicate that the effect of passive targeting by PEGylation and active targeting by peptides can be independent or combined, and that they should be evaluated on a case-by-case basis when designing new nanosystems for targeted therapies. Both myxoma peptide and cRGD should be considered for specific targeting to melanoma, but a thorough investigation of the cytotoxicity of the myxoma peptide to different cell lines remains to be performed.

Intratumoral gold-doxorubicin is effective in treating melanoma in mice.

Intratumoral injection of ultra-small gold nanoparticles (AuNPs) conjugated to doxorubicin (Au-Dox) is effective against both murine B16 and human SK-MEL-28 tumors in mice. Au-Dox suppresses growth of B16 tumors in immunocompetent mice by >70% for at least 19 days. In SK-MEL-28 xenografts, Au-Dox suppresses tumor growth almost completely for >13 weeks, while tumors treated with Dox alone demonstrate accelerated growth after 10 weeks. Histological analysis shows significant apoptosis and necrosis in Au-Dox treated tumors. Intratumoral injection is significantly more effective than intravenous injection, which leads to significant accumulation in liver and kidney with sub-therapeutic concentrations of Au-Dox. However, IV injection does not lead to significant damage in non-target organs, so improved targeting should permit this mode of delivery with little risk of systemic toxicity. The current construct is suitable for tumors accessible to intratumoral injection and represents a viable approach doxorubicin-resistant solid tumors. FROM THE CLINICAL EDITOR: Drug resistance is a significant problem in the fight against cancer. The authors describe a new approach in combating drug resistance in tumor cells by conjugating ultrasmall gold nanoparticles to doxorubicin. They tested the efficacy in in-vivo models using two melanoma cell lines. The promising results obtained from intra-tumoral injections contribute a way in future drug designs showing that conjugation to nanoparticles could lead to more effective and synergistic killing of tumor cells.

Immobilized phage proteins for specific detection of staphylococci.

Rapid, specific detection of pathogenic bacteria remains a major challenge in infectious disease diagnostics. Bacteriophages can show genus- or even species-level specificity and have been developed for biosensing purposes, but the possibility of using individual phage proteins for detection has not been fully explored. This work exploits the ability of specific phage proteins, the endolysins LysK and Φ11, and the bacteriocin lysostaphin, fixed on silicon wafers to bind staphylococci. The proteins show activity against eight tested clinical isolates of S. aureus and to S. epidermidis, but no binding to Escherichia coli and limited binding to Micrococcus. Binding was quantified by clearing assays in solution and by functionalization of silicon wafers followed by light microscopy. Bacterial binding densities on functionalized surfaces were ~3 cells/100 µm(2). The small size of the proteins makes the system robust and easy to handle, and the principle is generalizable to many different biosensor platforms, including label-free systems such as optical microresonators.

Photoluminescence of cerium fluoride and cerium-doped lanthanum fluoride nanoparticles and investigation of energy transfer to photosensitizer molecules.

CexLa1-xF3 nanoparticles have been proposed for use in nanoscintillator-photosensitizer systems, where excitation of nanoparticles by ionizing radiation would result in energy transfer to photosensitizer molecules, effectively combining the effects of radiotherapy and photodynamic therapy. Thus far, there have been few experimental investigations of such systems. This study reports novel synthesis methods for water-dispersible Ce0.1La0.9F3/LaF3 and CeF3/LaF3 core/shell nanoparticles and an investigation of energy transfer to photosensitizers. Unbound deuteroporphyrin IX 2,4-disulfonic acid was found to substantially quench the luminescence of large (>10 nm diameter) aminocaproic acid-stabilized nanoparticles at reasonable concentrations and loading amounts: up to 80% quenching at 6% w/w photosensitizer loading. Energy transfer was found to occur primarily through a cascade, with excitation of "regular" site Ce(3+) at 252 nm relayed to photosensitizer molecules at the nanoparticle surface through intermediate "perturbed" Ce(3+) sites. Smaller (<5 nm) citrate-stabilized nanoparticles were coated with the bisphosphonate alendronate, allowing covalent conjugation to chlorin e6 and resulting in static quenching of the nanoparticle luminescence: ∼50% at ∼0.44% w/w. These results provide insight into energy transfer mechanisms that may prove valuable for optimizing similar systems.

Cadmium sulfate and CdTe-quantum dots alter DNA repair in zebrafish (Danio rerio) liver cells.

Increasing use of quantum dots (QDs) makes it necessary to evaluate their toxicological impacts on aquatic organisms, since their contamination of surface water is inevitable. This study compares the genotoxic effects of ionic Cd versus CdTe nanocrystals in zebrafish hepatocytes. After 24h of CdSO4 or CdTe QD exposure, zebrafish liver (ZFL) cells showed a decreased number of viable cells, an accumulation of Cd, an increased formation of reactive oxygen species (ROS), and an induction of DNA strand breaks. Measured levels of stress defense and DNA repair genes were elevated in both cases. However, removal of bulky DNA adducts by nucleotide excision repair (NER) was inhibited with CdSO4 but not with CdTe QDs. The adverse effects caused by acute exposure of CdTe QDs might be mediated through differing mechanisms than those resulting from ionic cadmium toxicity, and studying the effects of metallic components may be not enough to explain QD toxicities in aquatic organisms.

Differential effects of ß-mercaptoethanol on CdSe/ZnS and InP/ZnS quantum dots.

The small thiol ß-mercaptoethanol (BME) has been used as an anti-blinking reagent for CdSe/ZnS quantum dots (QDs), although its effects on QD photoluminescence are complex. It acts as an antioxidant as well as a hole scavenger on both CdSe and CdTe, which leads to changes in emission intensity and lifetime that vary qualitatively according to BME concentration, time of incubation, and pH of the solution. Because the band edge energies of InP/ZnS are shifted from those of CdTe and CdSe, it may be expected that thiols including BME might be unable to trap holes from these QDs. In this study, we use steady-state and time-resolved emission spectroscopy with physical fitting models combined with blinking analysis to compare the effects of different concentrations of BME on CdSe/ZnS vs. InP/ZnS QDs over time. We also find excellent correspondence between simple physical model parameters and blinking off times, a finding that will be useful for all blinking studies involving semiconductor nanoparticles. BME alters blinking in InP/ZnS QDs with a single ZnS shell, but not those with double thickness shells. The effects are similar to those seen with CdSe/ZnS, despite very different effects of BME on steady-state spectra, and highly pH-dependent.

Recent advances in experimental design and data analysis to characterize prokaryotic motility.

Bacterial motility plays a key role in important cell processes such as chemotaxis and biofilm formation, but is challenging to quantify due to the small size of the individual microorganisms and the complex interplay of biological and physical factors that influence motility phenotypes. Swimming, the first type of motility described in bacteria, still remains largely unquantified. Light microscopy has enabled qualitative characterization of swimming patterns seen in different strains, such as run and tumble, run-reverse-flick, run and slow, stop and coil, and push and pull, which has allowed for elucidation of the underlying physics. However, quantifying these behaviors (e.g., identifying run distances and speeds, turn angles and behavior by surfaces or cell-cell interactions) remains a challenging task. A qualitative and quantitative understanding of bacterial motility is needed to bridge the gap between experimentation, omics analysis, and bacterial motility theory. In this review, we discuss the strengths and limitations of how phase contrast microscopy, fluorescence microscopy, and digital holographic microscopy have been used to quantify bacterial motility. Approaches to automated software analysis, including cell recognition, tracking, and track analysis, are also discussed with a view to providing a guide for experimenters to setting up the appropriate imaging and analysis system for their needs.

Quantification of Motility in Bacillus subtilis at Temperatures Up to 84°C Using a Submersible Volumetric Microscope and Automated Tracking.

We describe a system for high-temperature investigations of bacterial motility using a digital holographic microscope completely submerged in heated water. Temperatures above 90°C could be achieved, with a constant 5°C offset between the sample temperature and the surrounding water bath. Using this system, we observed active motility in Bacillus subtilis up to 66°C. As temperatures rose, most cells became immobilized on the surface, but a fraction of cells remained highly motile at distances of >100 µm above the surface. Suspended non-motile cells showed Brownian motion that scaled consistently with temperature and viscosity. A novel open-source automated tracking package was used to obtain 2D tracks of motile cells and quantify motility parameters, showing that swimming speed increased with temperature until ∼40°C, then plateaued. These findings are consistent with the observed heterogeneity of B. subtilis populations, and represent the highest reported temperature for swimming in this species. This technique is a simple, low-cost method for quantifying motility at high temperatures and could be useful for investigation of many different cell types, including thermophilic archaea.

Antimicrobial activity and cellular toxicity of nanoparticle-polymyxin B conjugates.

We investigate the antimicrobial activity and cytotoxicity to mammalian cells of conjugates of the peptide antibiotic polymyxin B (PMB) to Au nanoparticles and CdTe quantum dots. Au nanoparticles fully covered with PMB are identical in antimicrobial activity to the free drug alone, whereas partially-conjugated Au particles show decreased effectiveness in proportion to the concentration of Au. CdTe-PMB conjugates are more toxic to Escherichia coli than PMB alone, resulting in a flattening of the steep PMB dose-response curve. The effect is most pronounced at low concentrations of PMB, with a greater effect on the concentration required to reduce growth by half (IC50) than on the concentration needed to inhibit all growth (minimum inhibitory concentration, MIC). The Gram positive organism Staphylococcus aureus is resistant to both PMB and CdTe, showing minimal increased sensitivity when the two are conjugated. Measurement of reactive oxygen species (ROS) generation shows a significant reduction in photo-generated hydroxyl and superoxide radicals with CdTe-PMB as compared with bare CdTe. There is a corresponding reduction in toxicity of QD-PMB versus bare CdTe to mammalian cells, with nearly 100% survival in fibroblasts exposed to bactericidal concentrations of QD-PMB. The situation in bacteria is more complex: photoexcitation of the CdTe particles plays a small role in IC50 but has a significant effect on the MIC, suggesting that at least two different mechanisms are responsible for the antimicrobial action seen. These results show that it is possible to create antimicrobial agents using concentrations of CdTe quantum dots that do not harm mammalian cells.

Bacterial and mineral elements in an arctic biofilm: a correlative study using fluorescence and electron microscopy.

Few simple labeling methods exist for simultaneous fluorescence and electron microscopy of bacteria and biofilms. Here we describe the synthesis, characterization, and application of fluorescent nanoparticle quantum dot (QD) conjugates to target microbial species, including difficult to label Gram-negative strains. These QD conjugates impart contrast for both environmental scanning electron microscopy (ESEM) and fluorescence microscopy, permitting observation of living and fixed bacteria and biofilms. We apply these probes for studying biofilms extracted from perennial cold springs in the Canadian High Arctic, which is a particularly challenging system. In these biofilms, sulfur-metabolizing bacteria live in close association with unusual sulfur mineral formations. Following simple labeling protocols with the QD conjugates, we are able to image these organisms in fully-hydrated samples and visualize their relationship to the sulfur minerals using both ESEM and fluorescence microscopy. We then use scanning transmission electron microscopy to observe precipitated sulfur around individual cells and within the biofilm lattice. All combined, this information sheds light on the possible mechanisms of biofilm and mineral structure formation. These new QD conjugates and techniques are highly transferable to many other microbiological applications, especially those involving Gram-negative bacteria, and can be used for correlated fluorescence and electron microscopy.

Characterization of Retinol Stabilized in Phosphatidylcholine Vesicles with and without Antioxidants.

Retinol stability has been reported to be improved by encapsulation in liposomes, both with and without cholesterol. However, this improvement is limited because of lipid peroxidation. In this study, we compare the stability of retinol in phosphatidylcholine liposomes under ultraviolet (UV) light or standard room air, with and without the addition of antioxidants. Both butylated hydroxytoluene (BHT) and a proprietary mix (StoppOx) improved the shelf stability from <10 to over 30 d. The addition of cholesterol had no effect. Fluorescence imaging showed a heterogeneous distribution of retinol within the vesicles, including within the aqueous layer. Fluorescence lifetimes were equally heterogeneous. Under UV irradiation, StoppOx protected retinol for significantly longer than BHT and via different mechanisms. This suggests that natural antioxidants work well to improve the retinol stability, but that further work to determine the optimal vesicle structure remains to be performed.

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

The perennial springs at Gypsum Hill (GH) and Colour Peak (CP), situated at nearly 80 degrees N 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 degrees C to 6.9 degrees C, approximately 7.5% NaCl, 0-20 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 CO(2) under in situ conditions and a Thiomicrospira strain isolated from the streamers also actively oxidized sulfide and thiosulfate and fixed CO(2) 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, phototrophic-independent metabolism in a high Arctic winter environment characterized by air temperatures commonly below -40 degrees C and with an annual average air temperature of -15 degrees C. 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.

Scintillation Yield Estimates of Colloidal Cerium-Doped LaF3 Nanoparticles and Potential for "Deep PDT".

A hybrid of radiotherapy and photodynamic therapy (PDT) has been proposed in previously reported studies. This approach utilizes scintillating nanoparticles to transfer energy to attached photosensitizers, thus generating singlet oxygen for local killing of malignant cells. Its effectiveness strongly depends upon the scintillation yield of the nanoparticles. Using a liquid scintillator as a reference standard, we estimated the scintillation yield of Ce0.1La0.9F3/LaF3 core/shell nanoparticles at 28.9 mg/ml in water to be 350 photons/MeV under orthovoltage X-ray irradiation. The subsequent singlet oxygen production for a 60 Gy cumulative dose to cells was estimated to be four orders of magnitude lower than the "Niedre killing dose," used as a target value for effective cell killing. Without significant improvements in the radioluminescence properties of the nanoparticles, this approach to "deep PDT" is likely to be ineffective. Additional considerations and alternatives to singlet oxygen are discussed.

Standardizing the atomic description, axis and centre of biological ion channels.

A general representation of the atomic co-ordinates of a biological ion channel is obtained from a definition of channel axis and centre. Through rotation and translation of the channel, its centre becomes the origin of the standard co-ordinate system, and the channel axis becomes the system's z-axis. A method for determining the channel axis and centre based on the concepts of mass centre and mass moment of inertia is presented. The method for determining the channel axis can be directly applied to channels that adhere to two specific conditions regarding their geometry and mass distribution. Specific examples are given for Gramicidin A (GA), and the mammalian potassium channel Kv 1.2. For channels that do not adhere to these conditions, minor modifications of these procedures can be applied in determining the channel axis. Specific examples are given for the outer membrane bacterial porin OmpF, and for the staphylococcal pore-forming toxin alpha-hemolysin (alpha HL). The definitions and procedures presented are made in an effort to establish a standard basis for performing, sharing, and comparing computations in a consistent manner.