Although biological applications of quantum dots (QDs) by now are widely recognized and reported, routine production of small-size water-soluble and stable QDs still remains a challenge. In the present work we highlight prospects offered by the hydrothermal method to synthesize water-soluble QDs. We illustrate the method by synthesizing CdTe QDs which process some outstanding properties like highly luminescent red emission and high stability over a wide pH range. Compared with those synthesized via the traditional aqueous method, we show that CdTe QDs synthesized via the hydrothermal method feature higher photostability and lower cytotoxicity. Based on such prepared CdTe QDs, luminescent QD-IgG bioprobes were produced to detect the breast cancer marker Her2 on the surface of MCF-7 cancer cells, indicating that such prepared QD systems are promising candidates for use as bioprobes.
Blinking and time correlation between fluorescences of neighboring negatively charged CdSe/CdS and CdSe/ZnS colloidal quantum dots have been studied experimentally. A tendency of synchronous blinking, that is, a bunching effect, is clearly observed from two neighboring QDs with a spatial separation up to 1.1 mu m. We believe that our observations will help to better understand the mechanisms for the blinking.
A common method to assess the performance of (super resolution) microscopes is to use the localization precision of emitters as an estimate for the achieved resolution. Naturally, this is widely used in super resolution methods based on single molecule stochastic switching. This concept suffers from the fact that it is hard to calibrate measures against a real sample (a phantom), because true absolute positions of emitters are almost always unknown. For this reason, resolution estimates are potentially biased in an image since one is blind to true position accuracy, i.e. deviation in position measurement from true positions. We have solved this issue by imaging nanorods fabricated with DNA-origami. The nanorods used are designed to have emitters attached at each end in a well-defined and highly conserved distance. These structures are widely used to gauge localization precision. Here, we additionally determined the true achievable localization accuracy and compared this figure of merit to localization precision values for two common super resolution microscope methods STED and STORM.
The standard Ar ion laser in a confocal microscope was replaced with an intra-cavity frequency doubled Nd:YAG laser operating at 473 nm. The fluorescence image quality suggests that excitation at 473 nm could be preferable.
Before birth, the peripheral chemoreceptors located in the carotid bodies (CB) are adapted to the low fetal Po-2 and are relatively insensitive to hypoxia. After birth, the sensitivity of the CB to hypoxia is reset in response to the rise in Po-2. The mechanism underlying this resetting, which requires several days to complete, remains unknown. We have investigated the possibility that the hypoxia-inducible factors HIF-1 alpha and HIF-2 alpha, which are activated by oxygen deprivation, are involved in this resetting process. Accordingly, we used immunostaining and densitometry to quantitate the levels of the HIF-1 alpha and HIF-2 alpha proteins in the rat CB during early perinatal life and after exposure to in vivo hypoxia during adolescence. Tyrosine hydroxylase (TH) was used as a marker for catecholaminergic neurons and oxygen-sensitive cells in the CB. Double-immunostaining revealed constitutive expression of HIF-1 alpha in both glomus cells (TH+) and sustentacular cells (TH-) of the CB of adolescent rats. However, immunoreactivity toward HIF-2 alpha was restricted to glomus cells. After exposure to hypoxia (8% O-2, 6 h), the expression of HIF-1 alpha was selectively upregulated in glomus cells and apparent translocation of both HIF-1 alpha and HIF-2 alpha to the nucleus was observed. Both of these proteins were expressed constitutively in the CB during the perinatal transition period. During the first postnatal week, the intensity of immunostaining for HIF-1 alpha in glomus cells decreased markedly, whereas the level of HIF-2a remained constant. We suggest that this selective down-regulation of HIF-1 alpha may be involved in the postnatal maturation of CB responsiveness to hypoxia.
This paper presents a novel method for studying cilia forming cells in asymmetric microfluidic environments. It has previously been shown that bending the primary cilium by a fluid flow will give rise to a calcium signal, but the sensitivity for flow direction has so far not been studied. The microfluidic device presented here was designed for control of the local direction of fluid flow on the cellular level, and thus, enables studies of cellular response to a direction controlled cilium movement. Cells seeded on cover slips form cilia with the average length 2.9 μm after three days in culture and 4.3 μm after four days. Distinct calcium peaks were found after the initiation of flow in the channel. By using a microstructured flow system we have been able to study the sensitivity of confluent COS 7 cells expressing primary cilium to changes in fluid flow.
We present the first microfabricated microfluidic devices designed specifically for studies of primary cilium mediated cellular response to dynamic flow. The primary cilium functions as a mechano-sensor in renal tubular epithelium, sensing the extracellular fluid flow. Malfunction of cilia has been implicated in e.g. polycystic kidney disease and other pathological conditions. Bending of the primary cilium by fluid flow has been shown to give rise to an intracellular calcium signal, however little is known about the sensitivity to flow duration, magnitude and direction. This paper presents a novel method for studying cilia forming cells in asymmetric microfluidic environments. The microfluidic devices presented here were designed for a dynamic control of the local fluid flow on a cellular level, and thus, enables studies of cellular responses to an amplitude, frequency and direction controlled cilium movement.
The primary cilium is a ubiquitous organelle present on most mammalian cells. Malfunction of the organelle has been associated with various pathological disorders, many of which lead to cystic disorders in liver, pancreas, and kidney. Primary cilia have in kidney epithelial cells been observed to generate intracellular calcium in response to fluid flow, and disruption of proteins involved in this calcium signaling lead to autosomal dominant polycystic kidney disease, implying a direct connection between calcium signaling and cyst formation. It has also been shown that there is a significant lag between the onset of flow and initiation of the calcium signal. The present study focuses on the mechanics of cilium bending and the resulting calcium signal. Visualization of real-time cilium movements in response to different types of applied flow showed that the bending is fast compared with the initiation of calcium increase. Mathematical modeling of cilium and surrounding membrane was performed to deduce the relation between bending and membrane stress. The results showed a delay in stress buildup that was similar to the delay in calcium signal. Our results thus indicate that the delay in calcium response upon cilia bending is caused by mechanical properties of the cell membrane.
Speed is key during infectious disease outbreaks. It is essential, for example, to identify critical host binding factors to pathogens as fast as possible. The complexity of host plasma membrane is often a limiting factor hindering fast and accurate determination of host binding factors as well as high-throughput screening for neutralizing antimicrobial drug targets. Here, we describe a multiparametric and high-throughput platform tackling this bottleneck and enabling fast screens for host binding factors as well as new antiviral drug targets. The sensitivity and robustness of our platform were validated by blocking SARS-CoV-2 particles with nanobodies and IgGs from human serum samples.
Glutamate, by activating N-methyl-D-aspartate (NMDA) receptors, alters the balance between dopamine D1 and D2 receptor signaling, but the mechanism responsible for this effect has not been known. We report here, using immunocytochemistry of primary cultures of rat neostriatal neurons, that activation of NMDA receptors recruits D1 receptors from the interior of the cell to the plasma membrane while having no effect on the distribution of D2 receptors. The D1 receptors were concentrated in spines as shown by colocalization with phalloidin-labeled actin filaments. The effect of NMDA on D1 receptors was abolished by incubation of cells in calcium-free medium and was mimicked by the calcium ionophore lonomycin. Recruitment of D1 receptors from the interior of the cell to the membrane was confirmed by subcellular fractionation. The recruited D1 receptors were functional as demonstrated by an increase in dopamine-sensitive adenylyl cyclase activity in membranes derived from cells that had been pretreated with NMDA. These results provide evidence for regulated recruitment of a G protein-coupled receptor in neurons, provide a cell biological basis for the effect of NMDA on dopamine signaling, and reconcile the conflicting hyperdopaminergic and hypoglutamatergic hypotheses of schizophrenia.
The dopaminergic and glutamatergic systems interact to initiate and organize normal behavior, a communication that may be perturbed in many neuropsychiatric diseases, including schizophrenia. We show here that NMDA, by allosterically modifying NMDA receptors, can act as a scaffold to recruit laterally diffusing dopamine D1 receptors (D1R) to neuronal spines. Using organotypic culture from rat striatum transfected with D1R fused to a fluorescent protein, we show that the majority of dendritic D1R are in lateral diffusion and that their mobility is confined by interaction with NMDA receptors. Exposure to NMDA reduces the diffusion coefficient for D1R and causes an increase in the number of D1R-positive spines. Unexpectedly, the action of NMDA in potentiating D1R recruitment was independent of calcium flow via the NMDA receptor channel. Thus, a highly energy-efficient, diffusion-trap mechanism can account for intraneuronal interaction between the glutamatergic and dopaminergic systems and for regulation of the number of D1R-positive spines. This diffusion trap system represents a molecular mechanism for brain plasticity and offers a promising target for development of antipsychotic therapy
By devising careful electrophoresis, it was shown that at pH below 7.0, the electrophoretic mobility of 3-mercaptopropionic acid (3MPA) coated CdSe-ZnS core-shell quantum dots (denoted as QD-3MPA) was very small. At pH above 7.0, QD-3MPA migrated toward the anode, implying acid dissociation, and the degree of which was proportional to the pH value. QD-3MPA's electrophoretic mobility was impaired after adding sufficient Ca2+ ions to the QD solution and revived when a similar amount of Ca2+ chelators (ethylene glycol tetraacetic acid, EGTA) was added. This demonstrated that acid dissociation and its pH dependence of 3MPA on the QD surface are critical factors in understanding the electric and optical properties of QDs. The acid dissociated QD-3MPA interacted strongly with Ca2+, forming a charge neutral QD-3MPA Ca2+ complex in the absence of EGTA. First-principles study confirmed the observed experimental evidence. The strong ionic interaction between acid dissociated QD-3MPA and Ca2+ is critical for developing reliable QD-based biosensing assays. Moreover, the strategy and techniques reported in this work are easily applicable to other fluorescent biomarkers and therefore can be important for advancing in vivo and in vitro imaging, sensing, and labeling.
The task of separating an image into distinct components that represent different features plays an important role in many applications. Traditionally, such separation techniques are applied once the image in question has been reconstructed from measured data. We propose an efficient iterative algorithm, where reconstruction is performed jointly with the task of separation. A key assumption is that the image components have different sparse representations. The algorithm is based on a scheme that minimizes a functional composed of a data discrepancy term and the l(1)-norm of the coefficients of the different components with respect to their corresponding dictionaries. The performance is demonstrated for joint 2D deconvolution and separation into curve- and point-like components, and tests are performed on synthetic data as well as experimental stimulated emission depletion and confocal microscopy data. Experiments show that such a joint approach outperforms a sequential approach, where one first deconvolves data and then applies image separation.
The aim of this work was to study the dynamics of proteins near solid surfaces in the presence or absence of competing surfactants by means of total internal reflection fluorescence correlation spectroscopy (TIR-FCS). Two different proteins were studied, bovine serum albumin (BSA) and Thermomyces lanuginosus lipase (TLL). A nonionic/anionic (C12E6/LAS) surfactant composition was used to mimic a detergent formulation and the surfaces used were C 18 terminated glass. It was found that with increasing surfactant concentrations the term in the autocorrelation function (ACF) representing surface binding decreased. This Suggested that the proteins were competed off the hydrophobic surface by the surfactant. When fitting the measured ACF to a model for surface kinetics, it was seen that with raised C12E6/LAS concentration, the Surface interaction rate increased for both proteins. Under these experimental conditions this meant that the time the protein was bound to the surface decreased. At 10 mu M C12E6/LAS the surface interaction was not visible for BSA, whereas it was still distinguishable in the ACF for TLL. This indicated that TLL had a higher affinity than BSA for the C 18 surface. The study showed that TIR-FCS provides a useful tool to quantify the surfactant effect on proteins adsorption.
We have studied the mobility of active and inactive Thermomyces lanuginosus lipase (TLL) on a spin-coated trimyristin substrate surface using fluorescence recovery after photobleaching (FRAP) in a confocal microscopy setup. By photobleaching a circular spot of fluorescently labeled TLL adsorbed on a smooth trimyristin surface, both the diffusion coefficient D and the mobile fraction f could be quantified. FRAP was performed on surfaces with different surface density of lipase and as a function of time after adsorption. The data showed that the mobility of TLL was significantly higher on the trimyristin substrate surfaces compared to our previous studies on hydrophobic model surfaces. For both lipase variants, the diffusion decreased to similar rates at high relative surface density of lipase, suggesting that crowding effects are dominant with higher adsorbed amount of lipase. However, the diffusion coefficient at extrapolated infinite surface dilution, D-0, was higher for the active TLL compared to the inactive (D-0 = 17.9 x 10(-11) cm(2)/s vs D-0 = 4.1 x 10(-11) cm(2)/s, data for the first time interval after adsorption). Moreover, the diffusion decreased with time after adsorption, most evident for the active TLL. We explain the results by product inhibition, i.e., that the accumulation of negatively charged fatty acid products decreased the diffusion rate of active lipases with time. This was supported by sequential adsorption experiments, where the adsorbed amount under flow conditions was studied as a function of time after adsorption. A second injection of lipase led to a significantly lower increase in adsorbed amount when the trimyristin surface was pretreated with active TLL compared to pretreatment of inactive TLL.
This work was performed with the aim of comparing protein adsorption results obtained from the recently developed dual polarization interferometry (DPI) with the well-established surface plasmon resonance (SPR) technique. Both techniques use an evanescent field as the sensing element but completely different methods to calculate the adsorbed mass. As a test system we used adsorption of the lipase from Thermomyces lanuginosus (TLL) on C18 surfaces. The adsorbed amount calculated with both techniques is in good agreement, with both adsorption isotherms saturating at 1.30-1.35 mg/m(2) at TLL concentrations of 1000 nM and above. Therefore, this supports the use of both SPR and DPI as tools for studying protein adsorption, which is very important when comparing adsorption data obtained from the use different techniques. Due to the spot sensing in SPR, this technique is recommended for initial kinetic studies, whereas DPI is more accurate when the refractive index and thickness of the adsorbed layer is of more interest.
The adsorption and activity of Thermomyces lanuginosus lipase (TLL) was measured with dual polarization interferometry (DPI) and confocal microscopy at a hydrophilic and hydrophobic surface. In the adsorption isotherms, it was evident that TLL both had higher affinity for the hydrophobic surface and adsorbed to a higher adsorbed amount (1.90 mg/m(2)) compared to the hydrophilic surface (1.40-1.50 mg/m(2)). The thickness of the adsorbed layer was constant (similar to 3.5 nm) on both surfaces at an adsorbed amount > 1.0 mg/m(2), but decreased on the hydrophilic surface at lower surface coverage, which might be explained by partially unfolding of the TLL structure. However, a linear dependence of the refractive index of the adsorbed layer on adsorbed amount of TLL on C18 surfaces indicated that the structure of TLL was similar at low and high surface coverage. The activity of adsorbed TLL was measured towards carboxyfluorescein diacetate (CFDA) in solution, which upon lipase activity formed a fluorescent product. The surface fluorescence intensity increase was measured in a confocal microscope as a function of time after lipase adsorption. It was evident that TLL was more active on the hydrophilic surface, which suggested that a larger fraction of adsorbed TLL molecules were oriented with the active site facing the solution compared to the hydrophobic surface. Moreover, most of the activity remained when the TLL surface coverage decreased. Earlier reports on TLL surface mobility on the same surfaces have found that the lateral diffusion was highest on hydrophilic surfaces and at low surface coverage of TLL. Hence, a high lateral mobility might lead to a longer exposure time of the active site towards solution, thereby increasing the activity against a water-soluble substrate.
We have analyzed surface diffusion properties of a variant of Thermomyces lanuginosa lipase (TLL) on hydrophilic silica and silica methylated with dichlorodimethylsilane (DDS) or octadecyltrichlorosilane (OTS). For this study a novel method for analysis of diffusion on solid surfaces was developed. The method is based on fluorescence recovery after photobleaching using confocal microscopy. When a rectangular area of the sample was photobleached, fluorescence recovery could be analyzed as one-dimensional diffusion, resulting in simplified mathematical expressions for fitting the data. The method was initially tested by measuring bovine serum albumin diffusion on glass, which led to a diffusion coefficient in good correspondence to earlier reports. For the analysis of TLL diffusion, ellipsometry data of TLL adsorption were used to calibrate fluorescence intensity to surface density of lipase, enabling measurements of the diffusion coefficient at different surface densities. The average diffusion coefficient was calculated in two time intervals after adsorption. Mobile fraction and diffusion coefficient were lowest on the OTS surface, when extrapolated to infinite surface dilution. Moreover, the diffusion rate decreased with time on the hydrophobic surfaces. Our observations can be explained by the surface dependence on the distribution of orientations and conformations of adsorbed TLL, where the transition from the closed to the catalytically active open and more hydrophobic structure is important.
Detergency mechanisms of lipids from single cotton fibers were visualized by means of confocal laser scanning microscopy (CLSM). Fibers were soiled with different types of lipids: olive oil, lard and tri-C-10, and subsequently stained with the fluorescent probe Nile Red. A surfactant composition of 300 M C12E6/LAS (1:2 mol%) was used to mimic the surfactants used in a common washing solution. It was evident from the captured image series that the different kinds of soiling were removed by different mechanisms by the surfactants, depending on the fluidity of the lipid. Roll-up was the main mechanism when removing olive oil, whereas emulsification (necking) and/or solubilization were observed in the removal of lard and tri-C-10. Only 20-25% of the olive oil remained after treatment with surfactants, which was much less compared to the solid fats where roughly 50% remained at end of treatment. The effect of adding lipases to the detergent formulation was clearly seen, both by an apparently higher rate of removal of olive oil but also using double injection when removing lard. A first injection of only surfactants removed a certain part of the lard as emulsion droplets stuck onto the fiber. A second injection of both lipases and surfactants was able to remove some of the preformed emulsion particles and reduce the overall remaining lard content on the cotton fiber.
With the aim of being able to manipulate the processes involved in interfacial catalysis, we have studied the effects of a mixture of nonionic/anionic surfactants, C12E6/LAS (1: 2 mol %), on the adsorption and surface mobility of a lipase obtained from Thermomyces lanuginosus (TLL). Surface plasmon resonance (SPR) and ellipsometry were used to analyze the competitive adsorption process between surfactants and TLL onto hydrophobic model surfaces intended to mimic an oily substrate for the lipase. We obtained the surface diffusion coefficient of a fluorescently labeled TLL variant on silica silanized with octadecyltrichlorosilane (OTS) by fluorescence recovery after photobleaching (FRAP) on a confocal laser scanning microscope. By means of ellipsometry we calibrated the fluorescence intensity with the surface density of the lipase. The TLL diffusion was measured at different surface densities of the enzyme and at two time intervals after coadsorption with different concentrations of C12E6/LAS. The surfactant concentrations were chosen to represent concentrations below the critical micelle concentration (CMC), in the CMC region, and above the CMC. The apparent TLL surface diffusion was extrapolated to infinite surface dilution, D-0. We found that the presence of surfactants strongly modulated the surface mobility of TLL: with D-0 = 0.8 x 10(-11) cm(2)/s without surfactants and D-0 = 13.1 x 10(-11) cm(2)/s with surfactants above the CMC. The increase in lipase mobility on passing the CMC was also accompanied by a 2- fold increase in the mobile fraction of TLL. SPR analysis revealed that surface bound TLL was displaced by C12E6/LAS in a concentration-dependent manner, suggesting that the observed increase in surface mobility imparts bulk-mediated diffusion and so-called rebinding of TLL to the surface. Our combined results on lipase/surfactant competitive adsorption and lipase surface mobility show how surfactants may play an important role in regulating interfacial catalysis from physiological digestion to technical applications such as detergency.
The mobility of single lipase molecules has been analyzed using single molecule tracking on a trimyristin substrate surface. This was achieved by conjugating lipases to quantum dots and imaging on spin-coated trimyristin surfaces by means of confocal laser scanning microscopy. Image series of single lipase molecules were collected, and the diffusion coefficient was quantified by analyzing the mean square displacement of the calculated trajectories. During no-flow conditions, the lipase diffusion coefficient was (8.0 +/- 5.0) x 10(-10) cm(2)/s. The trajectories had a bead on a string appearance, with the lipase molecule restricted in certain regions of the surface and then migrating to another region where the restricted diffusion continued. This gave rise to clusters in the trajectories. When a flow was applied to the system, the total distance and average step length between the clusters increased, but the restricted diffusion in the cluster regions was unaffected. This can be explained by the lipase operating in two different modes on the surface. In the cluster regions, the lipase is likely oriented with the active site toward the surface and hydrolyzes the substrate. Between these regions, a diffusion process is proposed where the lipase is in contact with the surface but affected by the external flow.
Immunofluorescence microscopy is a valuable tool for analyzing protein expression and localization at a subcellular level thus providing information regarding protein function, interaction partners and its role in cellular processes. When performing sample fixation, parameters such as difference in accessibility of proteins present in various cellular compartments as well as the chemical composition of the protein to be studied, needs to be taken into account. However, in systematic and proteome-wide efforts, a need exists for standard fixation protocol(s) that works well for the majority of all proteins independent of subcellular localization. Here, we report on a study with the goal to find a standardized protocol based on the analysis of 18 human proteins localized in 11 different organelles and subcellular structures. Six fixation protocols were tested based on either dehydration by alcohols (methanol, ethanol or iso-propanol) or cross-linking by paraformaldehyde followed by detergent permeabilization (Triton X-100 or saponin) in three human cell lines. Our results show that cross-linking is essential for proteome-wide localization studies and that cross-linking using paraformaldehyde followed by Triton X-100 permeabilization successfully can be used as a single fixation protocol for systematic studies.
Pinopodes have been suggested to be markers of uterine receptivity. Heparin-binding epidermal growth factor-like growth factor (HB-EGF) is expressed in increasing amounts in the secretory phase endometrium and is considered to be important for the human implantation process. The aim of this study was to investigate a possible co-existence of pinopodes and HB-EGF in the normal human endometrium. Endometrial biopsies were obtained from women with normal menstrual cycles. The biopsies were examined by scanning electron microscopy for the detection of pinopodes, by immunohistochemistry for the expression of HB-EGF protein, and by confocal microscopy to determine if HB-EGF was present on the surface of the pinopodes. The expression of HB-EGF in luminal and glandular epithelium was highest when fully developed pinopodes were present. Using confocal microscopy it was shown that HB-EGF was present both inside the luminal epithelial cells and on the surface of pinopodes. These findings suggest that HB-EGF might play a role in both the attachment and penetration steps in the human implantation process. Furthermore, the immunohistochemical staining demonstrates that HB-EGF can be used as a marker for the implantation window.
With confocal microscopy new knowledge in cell physiology is acquireddaily. However, most cell assays today are carried out either as multiwellplate assays, or in standard petridish assays. These two methods havedifferent features and foci, but they have in common the large amount ofcells submitted for treatment and imaging. In order to study only a few cellson a more detailed level[1, 2] in a relevant context, we have designed, built,and evaluated a microfluidic system. It features 1) immobilization of cells inthree dimensions, 2) transportation of cell nutrients and treatments as wellas removal of residual products, 3) an extremely stable and physiologicallyrelevant gradient of chemical concentration distribution around the cell.Previous efforts in this field by our group revealed a few very importantissues, indicating that microfabrication would be the enabling technologyfor experiments on cells in asymmetricenvironments.
We present an improved device and method for studying cellular response onmicrofluidic controlled stimuli of primary cilia. The primary cilium functions as amechano-sensor in renal tubular epithelium. Malfunction of cilia has been implicated inpolycystic kidney decease as well as other kidney abnormalities. Bending of cilia willgive rise to an intracellular calcium signal [1,2,3], but little is known about theimportance of flow direction, magnitude and duration to the calcium response. Ourpreliminary results indicate flow speed sensitivity.
Spinal cord ependymal cells display neural stem cell properties in vitro and generate scar-forming astrocytes and remyelinating oligodendrocytes after injury. We report that ependymal cells are functionally heterogeneous and identify a small subpopulation (8% of ependymal cells and 0.1% of all cells in a spinal cord segment), which we denote ependymal A (EpA) cells, that accounts for the in vitro stem cell potential in the adult spinal cord. After spinal cord injury, EpA cells undergo self-renewing cell division as they give rise to differentiated progeny. Single-cell transcriptome analysis revealed a loss of ependymal cell gene expression programs as EpA cells gained signaling entropy and dedifferentiated to a stem-cell-like transcriptional state after an injury. We conclude that EpA cells are highly differentiated cells that can revert to a stem cell state and constitute a therapeutic target for spinal cord repair.
Lung epithelial cells are extensively exposed to nanoparticles present in the modern urban environment. Nanoparticles, including colloidal quantum dots (QDs), are also considered to be potentially useful carriers for the delivery of drugs into the body. It is therefore important to understand the ways of distribution and the effects of the various types of nanoparticles in the lung epithelium. We use a model system of liquid-covered human airway epithelial Calu-3 cell cultures to study the immediate and long-term effects of repeated deposition of colloidal 3-mercaptopropionic-acid coated CdSe-CdS/ZnS core-multishell QDs on the lung epithelial cell surface. By live confocal microscope imaging and by QD fluorescence measurements we show that the QD permeation through the mature epithelial monolayers is very limited. At the time of QD deposition, the transepithelial electrical resistance (TEER) of the epithelial monolayers transiently decreased, with the decrement being proportional to the QD dose. Repeated QD deposition, once every six days for two months, lead to accumulation of only small amounts of the QDs in the cell monolayer. However, it did not induce any noticeable changes in the long-term TEER and the molecular morphology of the cells. The colloidal 3-mercaptopropionic-acid coated CdSe-CdS/ZnS core-multishell QDs could therefore be potentially used for the delivery of drugs intended for the surface of the lung epithelia during limited treatment periods.
Pyelonephritis is one of the most common febrile diseases in children. If not treated appropriately, it causes irreversible renal damage and accounts for a large proportion of end stage renal failures(1). Renal scarring can occur in the absence of inflammatory cells, indicating that bacteria may have a direct signalling effect on renal cells(2). Intracellular calcium ([Ca2+](i)) oscillations can protect cells from the cytotoxic effects of prolonged increases in intracellular calcium(3,4). However, no pathophysiologically relevant protein that induces such oscillations has been identified. Here we show that infection by uropathogenic Escherichia coli induces a constant, low-frequency oscillatory [Ca2+](i) response in target primary rat renal epithelial cells induced by the secreted RTX (repeats-in-toxin) toxin alpha-haemolysin. The response depends on calcium influx through L-type calcium channels as well as from internal stores gated by inositol triphosphate. Internal calcium oscillations induced by alpha-haemolysin in a renal epithelial cell line stimulated production of cytokines interleukin (IL)-6 and IL-8. Our findings indicate a novel role for alpha-haemolysin in pyelonephritis: as an inducer of an oscillating second messenger response in target cells, which fine-tunes gene expression during the inflammatory response.
In recent years, many light-microscopy protocols have been published for visualization of nanoscale structures in the kidney. These protocols present researchers with new tools to evaluate both foot process anatomy and effacement, as well as protein distributions in foot processes, the slit diaphragm and in the glomerular basement membrane. However, these protocols either involve the application of different complicated super resolution microscopes or lengthy sample preparation protocols. Here, we present a fast and simple, five-hour long procedure for three-dimensional visualization of kidney morphology on all length scales. The protocol combines optical clearing and tissue expansion concepts to produce a mild swelling, sufficient for resolving nanoscale structures using a conventional confocal microscope. We show that the protocol can be applied to visualize a wide variety of pathologic features in both mouse and human kidneys. Thus, our fast and simple protocol can be beneficial for conventional microscopic evaluation of kidney tissue integrity both in research and possibly in future clinical routines.
The glomerular filtration barrier, consisting of podocyte foot processes with bridging slit diaphragm, glomerular basement membrane, and endothelium, is a key component for renal function. Previously, the subtlest elements of the filtration barrier have only been visualized using electron microscopy. However, electron microscopy is mostly restricted to ultrathin two-dimensional samples, and the possibility to simultaneously visualize multiple different proteins is limited. Therefore, we sought to implement a super-resolution immunofluorescence microscopy protocol for the study of the filtration barrier in the kidney. Recently, several optical clearing methods have been developed making it possible to image through large volumes of tissue and even whole organs using light microscopy. Here we found that hydrogel-based optical clearing is a beneficial tool to study intact renal tissue at the nanometer scale. When imaging samples using super-resolution STED microscopy, the staining quality was critical in order to assess correct nanoscale information. The signal-to-noise ratio and immunosignal homogeneity were both improved in optically cleared tissue. Thus, STED of slit diaphragms in fluorescently labelled optically cleared intact kidney samples is a new tool for studying the glomerular filtration barrier in health and disease.
Metabotropic glutamate receptor 5 (mGluR5) is highly expressed in the mammalian central nervous system (CNS). It is involved in multiple physiological functions and is a target for treatment of various CNS disorders, including schizophrenia. We report that Norbin, a neuron-specific protein, physically interacts with mGluR5 in vivo, increases the cell surface localization of the receptor, and positively regulates mGluR5 signaling. Genetic deletion of Norbin attenuates mGluR5-dependent stable changes in synaptic function measured as long-term depression or long-term potentiation of synaptic transmission in the hippocampus. As with mGluR5 knockout mice or mice treated with mGluR5-selective antagonists, Norbin knockout mice showed a behavioral phenotype associated with a rodent model of schizophrenia, as indexed by alterations both in sensorimotor gating and psychotomimetic-induced locomotor activity.
Background: Norbin is a neuron-specific, cytosolic protein that interacts with the metabotropic glutamate receptor 5 (mGluR5) and has a profound impact on mGluR5 signaling. Yet, little is known about its synaptic distribution. Results: Here we have analyzed the spatial relationship between Norbin, postsynaptic density protein 95 (PSD-95), actin and mGluR5 in spines using super-resolution microscopy. Norbin was found to have a high degree of colocalization with actin and a lower degree of colocalization with PSD-95. Co-immunoprecipitation studies confirmed that interaction occurs between Norbin and actin, but not between Norbin and PSD-95. Norbin was also found to have a high degree of colocalization with the perisynaptically located mGluR5. Findings based on structured illumination microscopy (3D-SIM) of exogenous expressed Norbin-GFP were confirmed by stimulated emission depletion microscopy (STED) of immunolabeled endogenous Norbin. Conclusions: Norbin associates with actin rather than with PSD-95 in dendritic spines. Results regarding protein localization and colocalization performed with conventional confocal microscopy must be interpreted with great caution. The now available super-resolution microscopy techniques provide more accurate information about sub-cellular protein localization than previously was possible.
In this tutorial review in the thematic series "Acoustofluidics", we discuss the implementation and practice of optical microscopy in acoustofluidic micro-devices. Examples are given from imaging of acoustophoretic manipulation of particles and cells in microfluidic channels, but most of the discussion is applicable to imaging in any lab-on-a-chip device. The discussion includes basic principles of optical microscopy, different microscopy modes and applications, and design criteria for micro-devices compatible with basic, as well as advanced, optical microscopy.
Nanoparticles of iron oxide generated by wearing of vehicles have been modelled with a tailored solution of size-uniform engineered magnetite particles produced by the Bradley reaction, a solvothermal metal-organic approach rendering hydrophilic particles. The latter does not bear any pronounced surface charge in analogy with that originating from anthropogenic sources in the environment. Physicochemical properties of the nanoparticles were thoroughly characterized by a wide range of methods, including XPD, TEM, SEM, DLS and spectroscopic techniques. The magnetite nanoparticles were found to be sensitive for transformation into maghemite under ambient conditions. This process was clearly revealed by Raman spectroscopy for high surface energy magnetite particles containing minor impurities of the hydromaghemite phase and was followed by quantitative measurements with EXAFS spectroscopy. In order to assess the toxicological effects of the produced nanoparticles in humans, with and without surface modification with ATP (a model of bio-corona formed in alveolar liquid), a pathway of potential uptake and clearance was modelled with a sequence of in vitro studies using A549 lung epithelial cells, lymphocyte 221-B cells, and 293T embryonal kidney cells, respectively. Raman microscopy unambiguously showed that magnetite nanoparticles are internalized within the A549 cells after 24 h co-incubation, and that the ATP ligand is retained on the nanoparticles throughout the uptake process. The toxicity of the nanoparticles was estimated using confocal fluorescence microscopy and indicated no principal difference for unmodified and modified particles, but revealed considerably different biochemical responses. The IL-8 cytokine response was found to be significantly lower for the magnetite nanoparticles compared to TiO2, while an enhancement of ROS was observed, which was further increased for the ATP-modified nanoparticles, implicating involvement of the ATP signalling pathway in the epithelium.
We carefully characterized the fluorescence blinking of single colloidal CdSe-CdS/ZnS core-multishell quantum dots (QDs) with different surface modifications, including octadecylamine (ODA) coated QDs dispersed in chloroform, aqueous 3-mercaptopropionic acids (3MPA) coated QDs in HEPES solution treated by Ca2+ ions and ethylene glycol tetraacetic acid (EGTA, Ca2+ chelator), and aqueous 3MPA-QDs treated by glycerol. It was found that the on- and off-state probability density distributions displayed different rules. The off-state probability density distributions of all QDs complied well with the inverse power law, while the on-state probability density distributions bended upwards in log-log scale, and the degree of the upwards-bending correlated strongly with QD surface modification and fluorescence brightness of the single QD. Further autocorrelation analysis revealed that the fluorescence time series of a single QD was more random when the single QD showed a stronger fluorescence. Realistic numerical simulations with input parameters from quantum mechanical calculations showed that the QD exciton was first generated by an excitation photon; It radiatively recombined to give QD's fluorescence response, i.e., the on-state, which displayed the upwards-bended on-state probability density distribution profile; The electron and/or the hole of the photoexcited exciton in the QD core, after tunneling to the QD surface, randomly walked through the two-dimensional network of the QD surface states, resulting in the off-state probability density distribution profile of the inverse power law. Surface modification modified the QD surface-state network, in turn modifying the on/off probability density distribution profiles. Our findings provide us with a novel highway of applying colloidal QDs to study microscopic physical, and chemical, processes in many fields including in vivo and in vitro imaging, sensing and labelling.