When light meets biology - how the specimen affects quantitative microscopy.

Fluorescence microscopy images should not be treated as perfect representations of biology. Many factors within the biospecimen itself can drastically affect quantitative microscopy data. Whereas some sample-specific considerations, such as photobleaching and autofluorescence, are more commonly discussed, a holistic discussion of sample-related issues (which includes less-routine topics such as quenching, scattering and biological anisotropy) is required to appropriately guide life scientists through the subtleties inherent to bioimaging. Here, we consider how the interplay between light and a sample can cause common experimental pitfalls and unanticipated errors when drawing biological conclusions. Although some of these discrepancies can be minimized or controlled for, others require more pragmatic considerations when interpreting image data. Ultimately, the power lies in the hands of the experimenter. The goal of this Review is therefore to survey how biological samples can skew quantification and interpretation of microscopy data. Furthermore, we offer a perspective on how to manage many of these potential pitfalls.

Probability of Immobilization on Host Cell Surface Regulates Viral Infectivity.

The efficiency of a virus to establish its infection in host cells varies broadly among viruses. It remains unclear if there is a key step in this process that controls viral infectivity. To address this question, we use single-particle tracking and Brownian dynamics simulation to examine human immunodeficiency virus type 1 (HIV-1) infection in cell culture. We find that the frequency of viral-cell encounters is consistent with diffusion-limited interactions. However, even under the most favorable conditions, only 1% of the viruses can become immobilized on cell surface and subsequently enter the cell. This is a result of weak interaction between viral surface gp120 and CD4 receptor, which is insufficient to form a stable complex the majority of the time. We provide the first direct quantitation for efficiencies of these events relevant to measured HIV-1 infectivity and demonstrate that immobilization on host cell surface post-virion-diffusion is the key step in viral infection. Variation of its probability controls the efficiency of a virus to infect its host cells. These results explain the low infectivity of cell-free HIV-1 in vitro and offer a potential rationale for the pervasive high efficiency of cell-to-cell transmission of animal viruses.

Orthophosphate Interactions with Destabilized PbO2 Scales.

This research presents two case studies in which a change in the disinfectant from free chlorine to chloramine caused an increase in lead corrosion. In both systems, the predominantly tetravalent lead (PbO2) scales destabilized as a result of disinfectant change. Orthophosphate corrosion control was used in both systems, and the effect of this treatment chemical on the destabilized PbO2 scales was examined. The absence of chemical reactivity between PbO2 and phosphorus is well known, and this research confirms that phosphorus does not interact with the legacy PbO2 scales. Instead, phosphorus and calcium were found to permeate through the destabilized PbO2 material and react with divalent lead [Pb(II)] at the surface of a basal litharge (PbO) layer. This reaction precipitated a crystalline lead phosphate in both systems, which could not be specifically identified by any known powder diffraction files. Further analysis suggested that the compound formed was not the typically modeled hydroxypyromorphite but rather a calcium-substituted hydroxypyromorphite. During scale formation, calcium is frequently bound to the Pb(II) phosphate crystal lattice structure, causing measurable crystal lattice distortion in powder X-ray diffraction patterns. The results of this study illustrate the longevity of legacy scales and how disequilibrium compounds persist long after treatment changes have been made.

Lead Particle Size Fractionation and Identification in Newark, New Jersey's Drinking Water.

Following a pH reduction in their drinking water over a span of more than 20 years, the City of Newark, New Jersey, has struggled with elevated lead (Pb) release from Pb service lines and domestic plumbing in the zone fed by the Pequannock Water Treatment Plant. In response, Newark initiated orthophosphate addition and provided faucet-mounted point-of-use (POU) filters and pitcher filters certified for Pb and particulate reduction under NSF/ANSI Standards 53 and 42 to residential homes in that zone. Water chemistry analysis and size fractionation sampling were performed at four of these houses. Analysis of the particulate material retained by the fractionation filters revealed that Pb was dominantly present in the water as fine Pb(II) orthophosphate particles. A considerable amount of the particulates occurred as a nanoscale fraction that sometimes passed through the POU faucet or pitcher filtration units. Scanning electron microscopy, transmission electron microscopy, and energy-dispersive spectroscopy analyses showed that the nanoparticles (<100 nm) and their aggregates were composed of Pb, phosphorus, and chlorine, which are consistent with pyromorphite, Pb5(PO4)3Cl. Electron diffraction and X-ray analyses supported the presence of hydroxypyromorphite and chloropyromorphite nanoparticles and the size range estimates from the imaging. This research confirmed that nonadherent Pb(II)-orthophosphate nanoparticles were an important form of Pb in drinking water in the Pequannock water quality zone of Newark.

Mineralogical Evidence of Galvanic Corrosion in Drinking Water Lead Pipe Joints.

Galvanic corrosion as a mechanism of toxic lead release into drinking water has been under scientific debate in the U.S. for over 30 years. Visual and mineralogical analysis of 28 lead pipe joints, excavated after 60+ years from eight U.S. water utilities, provided the first direct view of three distinct galvanic corrosion patterns in practice: (1) no evidence of galvanic corrosion; (2) galvanic corrosion with lead cathode; (3) galvanic corrosion with lead anode. Pattern 3 is consistent with empirical galvanic series (lead → brass → copper in order of increasing nobility) and poses the greatest risk of Pb exposure. Pattern 2 is consistent with galvanic battery reversion. The identification of copper-sulfate minerals (Pattern 2), and lead-sulfate and lead-chloride minerals (Pattern 3) in galvanic zones illustrated the migration of chloride and sulfate toward the anode. Geochemical modeling confirmed the required pH drop from the bulk water level to at least pH 3.0-4.0 (Pattern 2) and pH < 5.5 (Pattern 3) in order to form these minerals. Despite joints being over 60 years old, galvanic zones in Pattern 3 were active and possibly posed an important source of lead to drinking water. Importantly, Pattern 3 was not observed in samples from systems representing water qualities favoring PbO2 formation.

Quantitative Correlation between Infectivity and Gp120 Density on HIV-1 Virions Revealed by Optical Trapping Virometry.

The envelope glycoprotein (Env) gp120/gp41 is required for HIV-1 infection of host cells. Although in general it has been perceived that more Env gives rise to higher infectivity, the precise quantitative dependence of HIV-1 virion infectivity on Env density has remained unknown. Here we have developed a method to examine this dependence. This method involves 1) production of a set of single-cycle HIV-1 virions with varied density of Env on their surface, 2) site-specific labeling of Env-specific antibody Fab with a fluorophore at high efficiency, and 3) optical trapping virometry to measure the number of gp120 molecules on individual HIV-1 virions. The resulting gp120 density per virion is then correlated with the infectivity of the virions measured in cell culture. In the presence of DEAE-dextran, the polycation known to enhance HIV-1 infectivity in cell culture, virion infectivity follows gp120 density as a sigmoidal dependence and reaches an apparent plateau. This quantitative dependence can be described by a Hill equation, with a Hill coefficient of 2.4 ± 0.6. In contrast, in the absence of DEAE-dextran, virion infectivity increases monotonically with gp120 density and no saturation is observed under the experimental conditions. These results provide the first quantitative evidence that Env trimers cooperate on the virion surface to mediate productive infection by HIV-1. Moreover, as a result of the low number of Env trimers on individual virions, the number of additional Env trimers per virion that is required for the optimal infectivity will depend on the inclusion of facilitating agents during infection.

Multibuilding Block Janus Synthesized by Seed-Mediated Self-Assembly for Enhanced Photothermal Effects and Colored Brownian Motion in an Optical Trap.

The asymmetrical features and unique properties of multibuilding block Janus nanostructures (JNSs) provide superior functions for biomedical applications. However, their production process is very challenging. This problem has hampered the progress of JNS research and the exploration of their applications. In this study, an asymmetrical multibuilding block gold/iron oxide JNS has been generated to enhance photothermal effects and display colored Brownian motion in an optical trap. JNS is formed by seed-mediated self-assembly of nanoparticle-loaded thermocleavable micelles, where the hydrophobic backbones of the polymer are disrupted at high temperatures, resulting in secondary self-assembly and structural rearrangement. The JNS significantly enhances photothermal effects compared to their homogeneous counterpart after near-infrared (NIR) light irradiation. The asymmetrical distribution of gold and iron oxide within JNS also generates uneven thermophoretic force to display active colored Brownian rotational motion in a single-beam gradient optical trap. These properties indicate that the asymmetrical JNS could be employed as a strong photothermal therapy mediator and a fuel-free nanoscale Janus motor under NIR light.

Scale Formation Under Blended Phosphate Treatment for a Utility With Lead Pipes.

US corrosion control practice often assumes that the orthophosphate component of blended phosphate corrosion inhibitors causes the formation of low-solubility lead-orthophosphate solids that control lead release into drinking water. This study identified the solids that formed on the interior surface of a lead service line and a galvanized steel pipe excavated from a system using a proprietary blended phosphate chemical. The scale was analyzed by X-ray diffraction, X-ray fluorescence, and scanning electron microscopy/energy dispersive spectroscopy. Instead of crystalline lead-orthophosphate solids, a porous amorphous layer rich in aluminum, calcium, phosphorus, and lead was observed at the lead pipe scale-water interface. Thus, the mechanism inhibiting lead release into the water was not a thermodynamically predictable passivating lead-orthophosphate scale, but rather an amorphous barrier deposit that was possibly vulnerable to disturbances. Galvanized pipe scales showed relatively crystalline iron and zinc compounds, with additional surface deposition of aluminum, phosphorus, calcium, and lead.

Low contribution of PbO2-coated lead service lines to water lead contamination at the tap.

To determine if residential water sampling corroborates the expectation that formation of stable PbO2 coatings on lead service lines (LSLs) provides an effective lead release control strategy, lead profile sampling was evaluated for eight home kitchen taps in three U.S. cities with observed PbO2-coated LSLs (Newport, Rhode Island; Cincinnati and Oakwood, Ohio). After various water standing times, these LSLs typically released similar or lower peak lead levels (1 to 18 µg/L) than the lead levels from the respective kitchen faucets (1 to 130 µg/L), and frequently 50-80% lower than the lead levels typically reported from Pb(II)-coated LSLs in comparable published sampling studies. Prolonged stagnation (10-101 h) at the Cincinnati sites produced varying results. One site showed minimal (0-4 µg/L) increase in lead release from the PbO2-coated LSL, and persistence of free chlorine residual. However, the other site showed up to a 3-fold increase proportional to standing time, with essentially full depletion of the chlorine residual. Overall, lead release was consistently much lower than that reported in studies of Pb(II)-coated LSL scales, suggesting that natural formation of PbO2 in LSLs is an effective lead "corrosion" control strategy.

Plasticity-induced actin polymerization in the dendritic shaft regulates intracellular AMPA receptor trafficking.

AMPA-type receptors (AMPARs) are rapidly inserted into synapses undergoing plasticity to increase synaptic transmission, but it is not fully understood if and how AMPAR-containing vesicles are selectively trafficked to these synapses. Here, we developed a strategy to label AMPAR GluA1 subunits expressed from their endogenous loci in cultured rat hippocampal neurons and characterized the motion of GluA1-containing vesicles using single-particle tracking and mathematical modeling. We find that GluA1-containing vesicles are confined and concentrated near sites of stimulation-induced structural plasticity. We show that confinement is mediated by actin polymerization, which hinders the active transport of GluA1-containing vesicles along the length of the dendritic shaft by modulating the rheological properties of the cytoplasm. Actin polymerization also facilitates myosin-mediated transport of GluA1-containing vesicles to exocytic sites. We conclude that neurons utilize F-actin to increase vesicular GluA1 reservoirs and promote exocytosis proximal to the sites of synaptic activity.

Single-image diffusion coefficient measurements of proteins in free solution.

Diffusion coefficient measurements are important for many biological and material investigations, such as studies of particle dynamics and kinetics, and size determinations. Among current measurement methods, single particle tracking (SPT) offers the unique ability to simultaneously obtain location and diffusion information about a molecule while using only femtomoles of sample. However, the temporal resolution of SPT is limited to seconds for single-color-labeled samples. By directly imaging three-dimensional diffusing fluorescent proteins and studying the widths of their intensity profiles, we were able to determine the proteins' diffusion coefficients using single protein images of submillisecond exposure times. This simple method improves the temporal resolution of diffusion coefficient measurements to submilliseconds, and can be readily applied to a range of particle sizes in SPT investigations and applications in which diffusion coefficient measurements are needed, such as reaction kinetics and particle size determinations.

Single-image axial localization precision analysis for individual fluorophores.

Bio-mechanism investigations demand single particle tracking with high spatial and temporal resolutions which require single fluorophore 3D localization measurements with matching precision and speed. Although the precision for lateral-localization measurements is well described by an analytical expression, for the axial direction, it is often obtained by repeating location measurements or by estimating a lower bound. Here, we report a precision expression for an axial-localization method that analyzes the standard deviations of single fluorophores' intensity profiles. Like the lateral-localization precision, this expression includes all relevant experimental effects measurable from a gaussian intensity profile of the fluorophore. This expression completes the precision analysis for single-image 3D localization of individual fluorophores and lifts the temporal resolution to the typical exposure timescales of milliseconds.

A holistic approach to lead pipe scale analysis: Importance, methodology, and limitations.

With lead service lines (LSLs) remaining for decades to come, scale analyses are critical to helping limit lead exposure from drinking water. This laboratory has used an integrated suite of analytical techniques to characterize the elemental composition, mineral identification, and physical features of scales, helping the water industry to evaluate, predict, and reduce lead corrosion. The methods used in this laboratory to prepare and analyze the LSL scale, and guidance to achieving reliable and meaningful results, are described. Primary methods include the following: optical microscopy, powder X-ray diffraction, inductively coupled plasma spectroscopy, X-ray fluorescence, scanning electron microscopy with energy dispersive spectroscopy, combustion and coulometric analyses of C and S, and X-ray absorption spectroscopy. Examples of associated pitfalls and ways to avoid them are provided, including pipe excavation/transport, sample preparation, analysis, and data interpretation. Illustrative examples are presented of practical scale analysis questions that could be answered by combinations of pipe scale analyses.

Hexameric rings of the scaffolding protein CheW enhance response sensitivity and cooperativity in Escherichia coli chemoreceptor arrays.

The Escherichia coli chemoreceptor array is a supramolecular assembly that enables cells to respond to extracellular cues dynamically and with great precision and sensitivity. In the array, transmembrane receptors organized as trimers of dimers are connected at their cytoplasmic tips by hexameric rings of alternating subunits of the kinase CheA and the scaffolding protein CheW (CheA-CheW rings). Interactions of CheW molecules with the members of receptor trimers not directly bound to CheA-CheW rings may lead to the formation of hexameric CheW rings in the chemoreceptor array. Here, we detected such CheW rings with a cellular cysteine-directed cross-linking assay and explored the requirements for their formation and their participation in array assembly. We found that CheW ring formation varied with cellular CheW abundance, depended on the presence of receptors capable of a trimer-of-dimers arrangement, and did not require CheA. Cross-linking studies of a CheA~CheW fusion protein incapable of forming homomeric CheW oligomers demonstrated that CheW rings were not essential for the assembly of CheA-containing arrays. Förster resonance energy transfer (FRET)-based kinase assays of arrays containing variable amounts of CheW rings revealed that CheW rings enhanced the cooperativity and the sensitivity of the responses to attractants. We propose that six-membered CheW rings provide the additional interconnectivity required for optimal signaling and gradient tracking performance by chemosensory arrays.

Quantifying Molecular Dynamics within Complex Cellular Morphologies using LLSM-FRAP.

Quantifying molecular dynamics within the context of complex cellular morphologies is essential toward understanding the inner workings and function of cells. Fluorescence recovery after photobleaching (FRAP) is one of the most broadly applied techniques to measure the reaction diffusion dynamics of molecules in living cells. FRAP measurements typically restrict themselves to single-plane image acquisition within a subcellular-sized region of interest due to the limited temporal resolution and undesirable photobleaching induced by 3D fluorescence confocal or widefield microscopy. Here, an experimental and computational pipeline combining lattice light sheet microscopy, FRAP, and numerical simulations, offering rapid and minimally invasive quantification of molecular dynamics with respect to 3D cell morphology is presented. Having the opportunity to accurately measure and interpret the dynamics of molecules in 3D with respect to cell morphology has the potential to reveal unprecedented insights into the function of living cells.

Variability and sampling of lead (Pb) in drinking water: Assessing potential human exposure depends on the sampling protocol.

Lead (Pb) in drinking water has re-emerged as a modern public health threat which can vary widely in space and in time (i.e., between homes, within homes and even at the same tap over time). Spatial and temporal water Pb variability in buildings is the combined result of water chemistry, hydraulics, Pb plumbing materials and water use patterns. This makes it challenging to obtain meaningful water Pb data with which to estimate potential exposure to residents. The objectives of this review paper are to describe the root causes of intrinsic Pb variability in drinking water, which in turn impacts the numerous existing water sampling protocols for Pb. Such knowledge can assist the public health community, the drinking water industry, and other interested groups to interpret/compare existing drinking water Pb data, develop appropriate sampling protocols to answer specific questions relating to Pb in water, and understand potential exposure to Pb-contaminated water. Overall, review of the literature indicated that drinking water sampling for Pb assessment can serve many purposes. Regulatory compliance sampling protocols are useful in assessing community-wide compliance with a water Pb regulatory standard by typically employing practical single samples. More complex multi-sample protocols are useful for comprehensive Pb plumbing source determination (e.g., Pb service line, Pb brass faucet, Pb solder joint) or Pb form identification (i.e., particulate Pb release) in buildings. Exposure assessment sampling can employ cumulative water samples that directly capture an approximate average water Pb concentration over a prolonged period of normal household water use. Exposure assessment may conceivably also employ frequent random single samples, but this approach warrants further investigation. Each protocol has a specific use answering one or more questions relevant to Pb in water. In order to establish statistical correlations to blood Pb measurements or to predict blood Pb levels from existing datasets, the suitability of available drinking water Pb datasets in representing water Pb exposure needs to be understood and the uncertainties need to be characterized.

The LRRK2 G2019S mutation alters astrocyte-to-neuron communication via extracellular vesicles and induces neuron atrophy in a human iPSC-derived model of Parkinson's disease.

Astrocytes are essential cells of the central nervous system, characterized by dynamic relationships with neurons that range from functional metabolic interactions and regulation of neuronal firing activities, to the release of neurotrophic and neuroprotective factors. In Parkinson's disease (PD), dopaminergic neurons are progressively lost during the course of the disease, but the effects of PD on astrocytes and astrocyte-to-neuron communication remain largely unknown. This study focuses on the effects of the PD-related mutation LRRK2 G2019S in astrocytes generated from patient-derived induced pluripotent stem cells. We report the alteration of extracellular vesicle (EV) biogenesis in astrocytes and identify the abnormal accumulation of key PD-related proteins within multivesicular bodies (MVBs). We found that dopaminergic neurons internalize astrocyte-secreted EVs and that LRRK2 G2019S EVs are abnormally enriched in neurites and fail to provide full neurotrophic support to dopaminergic neurons. Thus, dysfunctional astrocyte-to-neuron communication via altered EV biological properties may participate in the progression of PD.

Single-image separation measurements of two unresolved fluorophores.

Measuring subdiffraction separations between single fluorescent particles is important for biological, nano-, and medical-technology studies. Major challenges include (i) measuring changing molecular separations with high temporal resolution while (ii) using identical fluorescent labels. Here we report a method that measures subdiffraction separations between two identical fluorophores by using a single image of milliseconds exposure time and a standard single-molecule fluorescent imaging setup. The fluorophores do not need to be bleached and the separations can be measured down to 40 nm with nanometer precision. The method is called single-molecule image deconvolution--SMID, and in this article it measures the standard deviation (SD) of Gaussian-approximated combined fluorescent intensity profiles of the two subdiffraction-separated fluorophores. This study enables measurements of (i) subdiffraction dimolecular separations using a single image, lifting the temporal resolution of seconds to milliseconds, while (ii) using identical fluorophores. The single-image nature of this dimer separation study makes it a single-image molecular analysis (SIMA) study.

Precision analysis for standard deviation measurements of immobile single fluorescent molecule images.

Standard deviation measurements of intensity profiles of stationary single fluorescent molecules are useful for studying axial localization, molecular orientation, and a fluorescence imaging system's spatial resolution. Here we report on the analysis of the precision of standard deviation measurements of intensity profiles of single fluorescent molecules imaged using an EMCCD camera.We have developed an analytical expression for the standard deviation measurement error of a single image which is a function of the total number of detected photons, the background photon noise, and the camera pixel size. The theoretical results agree well with the experimental, simulation, and numerical integration results. Using this expression, we show that single-molecule standard deviation measurements offer nanometer precision for a large range of experimental parameters.