Surface plasmon resonance imaging of excitable cells.

Surface plasmons (SPs) are surface charge density oscillations occuring at a metal/dieletric interface and are highly sensitive to refractive index variations adjacent to the surface. This sensitivity has been exploited successfully for chemical and biological assays. In these systems, a surface plasmon resonance (SPR)-based sensor detects temporal variations in the refractive index at a point. SPR has also been used in imaging systems where the spatial variations of refractive index in the sample provide the contrast mechanism. SPR imaging systems using high numerical aperture (NA) objective lenses have been designed to image adherent live cells with high magnification and near-diffraction limited spatial resolution. Addressing research questions in cell physiology and pharmacology often requires the development of a multimodal microscope where complementary information can be obtained. In this paper, we present the development of a multimodal microscope that combines SPR imaging with a number of additional imaging modalities including bright-field, epifluorescence, total internal reflection microscopy and SPR fluorescence microscopy. We used a high NA objective lens for SPR and TIR microscopy and the platform has been used to image live cell cultures demonstrating both fluorescent and label-free techniques. Both the SPR and TIR imaging systems feature a wide field of view (~300 µm) that allows measurements from multiple cells whilst maintaining a resolution sufficient to image fine cellular processes. The capability of the platform to perform label-free functional imaging of living cells was demonstrated by imaging the spatial variations in contractions from stem cell-derived cardiomyocytes. This technique shows promise for non-invasive imaging of cultured cells over very long periods of time during development.

Dynamic functional contribution of the water channel AQP5 to the water permeability of peripheral lens fiber cells.

Although the functionality of the lens water channels aquaporin 1 (AQP1; epithelium) and AQP0 (fiber cells) is well established, less is known about the role of AQP5 in the lens. Since in other tissues AQP5 functions as a regulated water channel with a water permeability (PH2O) some 20 times higher than AQP0, AQP5 could function to modulate PH2O in lens fiber cells. To test this possibility, a fluorescence dye dilution assay was used to calculate the relative PH2O of epithelial cells and fiber membrane vesicles isolated from either the mouse or rat lens, in the absence and presence of HgCl2, an inhibitor of AQP1 and AQP5. Immunolabeling of lens sections and fiber membrane vesicles from mouse and rat lenses revealed differences in the subcellular distributions of AQP5 in the outer cortex between species, with AQP5 being predominantly membranous in the mouse but predominantly cytoplasmic in the rat. In contrast, AQP0 labeling was always membranous in both species. This species-specific heterogeneity in AQP5 membrane localization was mirrored in measurements of PH2O, with only fiber membrane vesicles isolated from the mouse lens, exhibiting a significant Hg2+-sensitive contribution to PH2O. When rat lenses were first organ cultured, immunolabeling revealed an insertion of AQP5 into cortical fiber cells, and a significant increase in Hg2+-sensitive PH2O was detected in membrane vesicles. Our results show that AQP5 forms functional water channels in the rodent lens, and they suggest that dynamic membrane insertion of AQP5 may regulate water fluxes in the lens by modulating PH2O in the outer cortex.

Sensitive detection of voltage transients using differential intensity surface plasmon resonance system.

This paper describes theoretical and experimental study of the fundamentals of using surface plasmon resonance (SPR) for label-free detection of voltage. Plasmonic voltage sensing relies on the capacitive properties of metal-electrolyte interface that are governed by electrostatic interactions between charge carriers in both phases. Externally-applied voltage leads to changes in the free electron density in the surface of the metal, shifting the SPR position. The study shows the effects of the applied voltage on the shape of the SPR curve. It also provides a comparison between the theoretical and experimental response to the applied voltage. The response is presented in a universal term that can be used to assess the voltage sensitivity of different SPR instruments. Finally, it demonstrates the capacity of the SPR system in resolving dynamic voltage signals; a detection limit of 10mV with a temporal resolution of 5ms is achievable. These findings pave the way for the use of SPR systems in the detection of electrical activity of biological cells.

Thin-film optoacoustic transducers for subcellular Brillouin oscillation imaging of individual biological cells.

At low frequencies ultrasound is a valuable tool to mechanically characterize and image biological tissues. There is much interest in using high-frequency ultrasound to investigate single cells. Mechanical characterization of vegetal and biological cells by measurement of Brillouin oscillations has been demonstrated using ultrasound in the GHz range. This paper presents a method to extend this technique from the previously reported single-point measurements and line scans into a high-resolution acoustic imaging tool. Our technique uses a three-layered metal-dielectric-metal film as a transducer to launch acoustic waves into the cell we want to study. The design of this transducer and measuring system is optimized to overcome the vulnerability of a cell to the exposure of laser light and heat without sacrificing the signal-to-noise ratio. The transducer substrate shields the cell from the laser radiation, efficiently generates acoustic waves, facilitates optical detection in transmission, and aids with heat dissipation away from the cell. This paper discusses the design of the transducers and instrumentation and presents Brillouin frequency images on phantom, fixed, and living cells.

Functional gap junctions accumulate at the immunological synapse and contribute to T cell activation.

Gap junction (GJ) mediates intercellular communication through linked hemichannels from each of two adjacent cells. Using human and mouse models, we show that connexin 43 (Cx43), the main GJ protein in the immune system, was recruited to the immunological synapse during T cell priming as both GJs and stand-alone hemichannels. Cx43 accumulation at the synapse was Ag specific and time dependent, and required an intact actin cytoskeleton. Fluorescence recovery after photobleaching and Cx43-specific inhibitors were used to prove that intercellular communication between T cells and dendritic cells is bidirectional and specifically mediated by Cx43. Moreover, this intercellular cross talk contributed to T cell activation as silencing of Cx43 with an antisense or inhibition of GJ docking impaired intracellular Ca(2+) responses and cytokine release by T cells. These findings identify Cx43 as an important functional component of the immunological synapse and reveal a crucial role for GJs and hemichannels as coordinators of the dendritic cell-T cell signaling machinery that regulates T cell activation.

The Modular µSiM: A Mass Produced, Rapidly Assembled, and Reconfigurable Platform for the Study of Barrier Tissue Models In Vitro.

Advanced in vitro tissue chip models can reduce and replace animal experimentation and may eventually support "on-chip" clinical trials. To realize this potential, however, tissue chip platforms must be both mass-produced and reconfigurable to allow for customized design. To address these unmet needs, an extension of the µSiM (microdevice featuring a silicon-nitride membrane) platform is introduced. The modular µSiM (m-µSiM) uses mass-produced components to enable rapid assembly and reconfiguration by laboratories without knowledge of microfabrication. The utility of the m-µSiM is demonstrated by establishing an hiPSC-derived blood-brain barrier (BBB) in bioengineering and nonengineering, brain barriers focused laboratories. In situ and sampling-based assays of small molecule diffusion are developed and validated as a measure of barrier function. BBB properties show excellent interlaboratory agreement and match expectations from literature, validating the m-µSiM as a platform for barrier models and demonstrating successful dissemination of components and protocols. The ability to quickly reconfigure the m-µSiM for coculture and immune cell transmigration studies through addition of accessories and/or quick exchange of components is then demonstrated. Because the development of modified components and accessories is easily achieved, custom designs of the m-µSiM shall be accessible to any laboratory desiring a barrier-style tissue chip platform.

Non-invasive hydrodynamic imaging in plant roots at cellular resolution.

A key impediment to studying water-related mechanisms in plants is the inability to non-invasively image water fluxes in cells at high temporal and spatial resolution. Here, we report that Raman microspectroscopy, complemented by hydrodynamic modelling, can achieve this goal - monitoring hydrodynamics within living root tissues at cell- and sub-second-scale resolutions. Raman imaging of water-transporting xylem vessels in Arabidopsis thaliana mutant roots reveals faster xylem water transport in endodermal diffusion barrier mutants. Furthermore, transverse line scans across the root suggest water transported via the root xylem does not re-enter outer root tissues nor the surrounding soil when en-route to shoot tissues if endodermal diffusion barriers are intact, thereby separating 'two water worlds'.

Effects of Toxoplasma gondii infection on the function and integrity of human cerebrovascular endothelial cells and the influence of verapamil treatment in vitro.

Toxoplasma gondii can cause parasitic encephalitis, a life-threatening infection that predominately occurs in immunocompromised individuals. T. gondii has the ability to invade the brain, but the mechanisms by which this parasite crosses the blood-brain-barrier (BBB) remain incompletely understood. The present study reports the changes associated with infection and replication of T. gondii within human brain microvascular endothelial cells (BMECs) in vitro. Our results indicated that exposure to T. gondii had an adverse impact on the function and integrity of the BMECs - through induction of cell cycle arrest, disruption of the BMEC barrier integrity, reduction of cellular viability and vitality, depolarization of the mitochondrial membrane potential, increase of the DNA fragmentation, and alteration of the expression of immune response and tight junction genes. The calcium channel/P-glycoprotein transporter inhibitor verapamil was effective in inhibiting T. gondii crossing the BMECs in a dose-dependent manner. The present study showed that T. gondii can compromise several functions of BMECs and demonstrated the ability of verapamil to inhibit T. gondii crossing of the BMECs in vitro.

Regulation of lens volume: implications for lens transparency.

Lens transparency is critically dependent on the maintenance of an ordered tissue architecture, and disruption of this order leads to light scatter and eventually lens cataract. Hence the volume of the fiber cells that make up the bulk of the lens needs to be tightly regulated if lens transparency is to be preserved. While it has long been appreciated that the lens can regulate its volume when placed in anisosmotic solutions, recent work suggests that the lens also actively maintains its volume under steady-state conditions. Furthermore, the process of fiber cell elongation necessitates that differentiating fiber cells dramatically increase their volume in response to growth factors. The cellular transport mechanisms that mediate the regulation of fiber cell volume in the lens cortex are only just beginning to be elucidated. In this region, fiber cells are continuously undergoing a process of differentiation that creates an inherent gradient of cells at different stages of elongation. These cells express different complements of transport proteins involved in volume regulation. In addition, transport processes at different depths into the lens are differentially influenced by electrochemical gradients that alter with distance into the lens. Taken together, our work suggests that the lens has spatially distinct ion influx and efflux pathways that interact to control its steady-state volume, its response to hypotonic swelling, and the elongation of differentiating fibers. Based on this work, we present a model which may explain the unique damage phenotype observed in diabetic cataract, in terms of the uncoupling or dysregulation of these ion influx and efflux pathways.

Molecular and functional mapping of regional differences in P2Y receptor expression in the rat lens.

Extracellular ATP has been shown to mobilize intracellular Ca(2+) in cultured ovine lens epithelial cells and in human lens epithelium, suggesting a role for purines in the modulation of lens transparency. In this study, we characterized the expression profiles of P2Y receptor isoforms throughout the rat lens at both the molecular and the functional levels. RT-PCR indicated that P2Y(1), P2Y(2), P2Y(4) and P2Y(6) are expressed in the lens, while P2Y(12), P2Y(13) and P2Y(14) are not. Immunohistochemistry, using isoform specific antibodies, indicated that the epithelium does not express P2Y(1) and P2Y(2), but that the underlying fiber cells, which differentiate from the epithelial cells, exhibit strong membranous labeling. Although co-expressed in fiber cells, differences in P2Y(1) and P2Y(2) expression were apparent. P2Y(1) expression extended deeper into the lens than P2Y(2), and its expression co-localized with Cx50 gap junction plaques, while P2Y(2) did not. Labeling for P2Y(4) and P2Y(6) receptors were observed in both epithelial cells and fiber cells, but the labeling was predominantly cytoplasmic in nature. While purine agonist (ATP, ADP, UTP and UDP) application to the lens induced mobilization of intracellular Ca(2+) in cortical fiber cells, little to no effect was observed in the anterior and equatorial epithelium. Thus the inability of UTP and UDP to mobilize intracellular Ca(2+) in the epithelium and the predominately cytoplasmic location of P2Y(4) and P2Y(6) suggests that these receptors may represent an inactive pool of receptors that may be activated under non-physiological conditions. In contrast, our results indicated that P2Y(1) and P2Y(2) are functionally active in fiber cells and their differential subcellular expression patterns suggest they may regulate distinct processes in the lens under steady state conditions.

TEM Tomography of Pores with Application to Computational Nanoscale Flows in Nanoporous Silicon Nitride (NPN).

Silicon nanomembrane technologies (NPN, pnc-Si, and others) have been used commercially as electron microscopy (EM) substrates, and as filters with nanometer-resolution size cut-offs. Combined with EM, these materials provide a platform for catching or suspending nanoscale-size structures for analysis. Usefully, the nanomembrane itself can be manufactured to achieve a variety of nanopore topographies. The size, shapes, and surfaces of nanopores will influence transport, fouling, sieving, and electrical behavior. Electron tomography (ET) techniques used to recreate nanoscale-sized structures would provide an excellent way to capture this variation. Therefore, we modified a sample holder to accept our standardized 5.4 mm × 5.4 mm silicon nanomembrane chips and imaged NPN nanomembranes (50⁻100 nm thick, 10⁻100 nm nanopore diameters) using transmission electron microscopy (TEM). After imaging and ET reconstruction using a series of freely available tools (ImageJ, TomoJ, SEG3D2, Meshlab), we used COMSOL Multiphysics™ to simulate fluid flow inside a reconstructed nanopore. The results show flow profiles with significantly more complexity than a simple cylindrical model would predict, with regions of stagnation inside the nanopores. We expect that such tomographic reconstructions of ultrathin nanopores will be valuable in elucidating the physics that underlie the many applications of silicon nanomembranes.

Rapid one-step biotinylation of biological and non-biological surfaces.

We describe a rapid one-step method to biotinylate virtually any biological or non-biological surface. Contacting a solution of biotin-spacer-lipid constructs with a surface will form a coating within seconds on non-biological surfaces or within minutes on most biological membranes including membrane viruses. The resultant biotinylated surface can then be used to interact with avidinylated conjugates, beads, vesicles, surfaces or cells.

High resolution 3D imaging of living cells with sub-optical wavelength phonons.

Label-free imaging of living cells below the optical diffraction limit poses great challenges for optical microscopy. Biologically relevant structural information remains below the Rayleigh limit and beyond the reach of conventional microscopes. Super-resolution techniques are typically based on the non-linear and stochastic response of fluorescent labels which can be toxic and interfere with cell function. In this paper we present, for the first time, imaging of live cells using sub-optical wavelength phonons. The axial imaging resolution of our system is determined by the acoustic wavelength (λa = λprobe/2n) and not on the NA of the optics allowing sub-optical wavelength acoustic sectioning of samples using the time of flight. The transverse resolution is currently limited to the optical spot size. The contrast mechanism is significantly determined by the mechanical properties of the cells and requires no additional contrast agent, stain or label to image the cell structure. The ability to breach the optical diffraction limit to image living cells acoustically promises to bring a new suite of imaging technologies to bear in answering exigent questions in cell biology and biomedicine.

Cl- influx into rat cortical lens fiber cells is mediated by a Cl- conductance that is not ClC-2 or -3.

PURPOSE: Exposure of organ-cultured lenses to Cl(-) channel blockers under isotonic conditions induces a localized cortical zone of extracellular space dilations. The purpose of this study was to investigate whether elongated lens fiber cells from this zone contain an anion conductance that mediates Cl(-) influx and whether two chloride channel isoforms known to be expressed in the lens (ClC-2 and -3) are responsible. METHODS: Fiber cells were isolated by enzymatic dissociation in the presence of Gd(3+) and Co(2+) and their electrical properties analyzed by whole-cell patch clamping. Cells from the zone of extracellular space dilations were selected for analysis on the basis of cell length. RT-PCR and immunocytochemistry were used to determine whether ClC-2 or -3 channel isoforms are expressed in fiber cells located in the zone of extracellular space dilations. RESULTS: Cells from the zone of extracellular space dilations were typically >120 microm in length and exhibited an outwardly rectifying Cl(-) conductance that was blocked by DIDS (4,4'-diisothiocyanostilbene-2,2'-disulfonic acid) and displayed an anion selectivity sequence of I(-) > Cl(-) >> gluconate. ClC-2 and -3 were found to be expressed at the transcript and protein level in lens fiber cells, but subsequent immunocytochemical studies indicated that expressed proteins did not colocalize with cell membranes in the zone of extracellular space dilations, being predominately cytoplasmic in nature. CONCLUSIONS: Taken together, the data indicate that extracellular space dilations are due to the inhibition of a Cl(-) channel(s) that normally mediates Cl(-) influx into cortical lens fiber cells under isotonic conditions. The molecular identity of this channel remains to be determined.

Whole-cell patch clamping of isolated fiber cells confirms that spatially distinct Cl- influx and efflux pathways exist in the cortex of the rat lens.

PURPOSE: To test the hypothesis that lens fiber cells use different combinations of transport proteins to mediate Cl influx and efflux in order to regulate their steady state volume. METHODS: Cells were isolated from rat lenses by enzymatic dissociation in the presence of Gd(3+), and short and long fiber cells were assigned to peripheral efflux and deeper influx zones, respectively. Electrical properties were of isolated cells, and whole lenses were analyzed by using whole-cell patch clamping and intracellular microelectrodes, respectively, before and after exposure to hyposmotic challenge and/or the addition of [(dihydronindenyl)oxy] alkanoic acid (DIOA). RESULTS: Cells from the influx zone were dominated by an outwardly rectifying Cl(-) conductance, and exposure to hyposmotic challenge increased this conductance. Cells isolated from the efflux zone were dominated by K(+) conductance(s) with only a minimal contribution from the Cl(-) conductance. Exposure of cells that exhibited a minimal baseline Cl(-) conductance to hyposmotic challenge caused swelling and a transient increase in Cl(-) current. In other cells that initially lacked a Cl(-) conductance, hyposmotic challenge caused swelling, but no increase in outward current. However, the subsequent addition of DIOA exacerbated swelling and activated a Cl(-) current. Under isosmotic conditions, addition of DIOA also induced cell swelling and the transient activation of a Cl(-) current. In whole lenses, exposure to hyposmotic challenge increased the contribution of an anion conductance to the membrane potential. CONCLUSIONS: In peripheral cells, Cl(-) efflux is primarily mediated by potassium chloride cotransporters (KCCs) and its activity can be upregulated by hyposmotic challenge. In addition, these cells also contain a Cl(-) channel that exhibits a variable baseline activity level and that can be recruited to effect regulatory volume decrease if the KCC transporters are inhibited.

Differentiation-dependent changes in the membrane properties of fiber cells isolated from the rat lens.

Impedance measurements in whole lenses showed that lens fiber cells possess different permeability properties to the epithelial cells from which they differentiate. To confirm these observations at the cellular level, we analyzed the membrane properties of fiber cells isolated in the presence of the nonselective cation channel inhibitor Gd3+. Isolated fiber cells were viable in physiological [Ca2+] and exhibited a range of lengths that reflected their stage of differentiation. Analysis of a large population of fiber cells revealed a subgroup of cells whose conductivity matched values measured in the whole lens (1). In this group of cells, membrane resistance, conductivity, and reversal potential all varied with cell length, suggesting that the process of differentiation is associated with a change in the membrane properties of fiber cells. Using pharmacology and ion substitution experiments, we showed that newly differentiated fiber cells (<150 microm) contained variable combinations of Ba2+-and tetraethylammonium-sensitive K+ currents. Longer fiber cells (150-650 microm) were dominated by a lyotropic anion conductance, which also appears to plays a role in the intact lens. Longer cells also exhibited a low-level, nonselective conductance that was eliminated by the replacement of extracellular Na+ with N-methyl-d-glucamine, indicating that the lens contains both Gd3+-sensitive and -insensitive nonselective cation conductances. Fiber cell differentiation is therefore associated with a shift in membrane permeability from a dominant K+ conductance(s) toward larger contributions from anion and nonselective cation conductances as fiber cells elongate.

Multiphoton imaging of chick retinal development in relation to gap junctional communication.

Neural progenitor cells in the developing retina extend processes that stretch from the basal vitread surface to the apical ventricular surface. During the cell cycle, the nucleus undergoes interkinetic nuclear migration (INM), moving in a vitread direction during G1, passing through S-phase at its peak and then, on entering G2, returning towards the ventricular surface where it enters M-phase and divides. We have previously shown that individual saltatory movements of the nucleus correlate with transient changes in cytosolic calcium concentration within these progenitor cells and that these events spread to neighbouring progenitors through connexin43 (Cx43) gap junction channels, thereby coordinating the migration of coupled clusters of cells. Disrupting coupling with pharmacological agents, Cx43-specific antisense oligodeoxynucleotides (asODNs) or dominant negative Cx43 (dnCx43) inhibits the sharing of calcium events, reducing the number that each cell experiences and significantly slowing INM. We have developed protocols for imaging migrating progenitor cells by confocal microscopy over relatively short periods, and by multiphoton microscopy over more extended periods that include complete cell cycles. We find that perturbing gap junctional communication not only slows the INM of progenitor cells but also apparently prevents them from changing direction at critical phases of the cell cycle. It also disrupts the migration of young neurons to their appropriate layers after terminal division and leads to their ectopic differentiation. The ability to perform extended time-lapse imaging over 3D volumes in living retina using multiphoton microscopy should now allow fundamental mechanisms governing development of the retinal neuroepithelium to be probed in detail.