Epifluorescence Imaging Research Articles

Alcohol-Induced Protein Kinase Cα Phosphorylation of Munc18c in Carbachol-Stimulated Acini Causes Basolateral Exocytosis

Acute or chronic alcohol treatment does little to the exocrine pancreas but predisposes the pancreas to postprandial cholinergic stimulation that triggers cellular events leading to pancreatitis. This alcohol-induced susceptibility mechanism of pancreatitis is unknown. We employed alcohol-treated dispersed rat pancreatic acini and alcohol diet-fed rats to examine the effects of submaximal carbachol-induced changes in exocytosis (FM1-43 epifluorescence imaging and electron microscopy), Munc18c cellular translocation (confocal microscopy and subcellular fractionation), and protein kinase C (PKC) alpha-induced phosphorylation in relation to pancreatitis. Acute low-dose alcohol (20 mmol/L) in vitro exposure or chronic alcohol diet reduces postprandial cholinergic-stimulated amylase secretion from rat pancreatic acinar cells by blocking apical exocytosis and redirecting exocytosis to less efficient basolateral plasma membrane sites. This ectopic exocytosis is mediated by PKCalpha-induced phosphorylation of Munc18c, causing Munc18c displacement from the basolateral plasma membrane into the cytosol in which it undergoes proteolytic degradation; these processes can be blocked by PKCalpha inhibition. We conclude that sequential low-dose alcohol and postprandial cholinergic stimulation can induce PKCalpha-mediated Munc18c plasma membrane displacement. This relieves cognate SNARE proteins on zymogen granules and basolateral membrane to complex and consummate pathologic ectopic exocytosis at the basolateral surface. This change in vesicle trafficking may be related to the pathogenesis of pancreatitis.

Epifluorescence microscopy and image analysis of high‐level polycyclic aromatic hydrocarbon contamination in soils

Interactions between polycyclic aromatic hydrocarbons (PAHs) and soil are an important determinant of their chemical availability and transport. Laboratory examination of microscale PAH-soil interaction is limited by the availability of methods for particle-scale observation. Inverted epifluorescence microscopy, combined with digital photography and computer image analysis, was evaluated for specificity and linearity using dissolved PAHs. A pyrene filter (excitation wavelength, 360-400 nm; emission wavelength, 450-520 nm) gave nonspecific PAH fluorescence, and bias for fluoranthene, benzo[b]fluoranthene, benzo[g,h,i]perylene, and benz[a]anthracene was quantified in comparison to that for pyrene. Concentrations ranging from 1 to 10 mM for anthracene, fluoranthene, and pyrene and from 1 to 50 mM for naphthalene produced a linear response with low interpixel variability. Liquid-phase analyses validated use of the technique for the descriptive analysis of PAH distribution in solid samples, but liquid-phase calibration was not quantitative for spiked or field-contaminated soils. The mean luminance for three field soils was proportional to the values predicted from their chemically measured concentrations and to values from spiked, aged, uncontaminated materials. Image analysis of laboratory- and field-contaminated samples determined the area distribution of fluorescent intensity and the size of fluorescent areas exceeding a threshold luminance. These qualitative descriptions of the microscale spatial distribution of PAH contamination are presented as potential endpoints for future research on biogeochemical interactions in heavily contaminated solids.

GLUT4 is internalized by a cholesterol-dependent nystatin-sensitive mechanism inhibited by insulin

Insulin slows GLUT4 internalization by an unknown mechanism. Here we show that in unstimulated adipocytes, GLUT4 is internalized by two mechanisms. Approximately 80% of GLUT4 is internalized by a mechanism that is sensitive to the cholesterol-aggregating drug nystatin, and is independent of AP-2 clathrin adaptor and two putative GLUT4 endocytic motifs. The remaining GLUT4 is internalized by an AP-2-dependent, nystatin-resistant pathway that requires the FQQI GLUT4 motif. Insulin inhibits GLUT4 uptake by the nystatin-sensitive pathway and, consequently, GLUT4 is internalized by the AP-2-dependent pathway in stimulated adipocytes. The phenylalanine-based FQQI GLUT4 motif promotes AP-2-dependent internalization less rapidly than a tyrosine-based motif, the classic form of aromatic-based motifs. Thus, both a change in the predominant endocytosis pathway and the specific use of a suboptimal internalization motif contribute to the slowing of GLUT4 internalization in insulin-stimulated adipocytes. Insulin also inhibits the uptake of cholera-toxin B, indicating that insulin broadly regulates cholesterol-dependent uptake mechanisms rather than specially targeting GLUT4. Our work thus identifies cholesterol-dependent uptake as a novel target of insulin action in adipocytes.

Epi-Fluorescence Imaging of Colloid Transport in Porous Media at Decimeter Scales

A noninvasive epi-fluorescence imaging technique was developed for real-time observation of colloid transport in porous media at decimeter scales. Fluorescent latex microspheres and translucent quartz sand were used as a model colloid-porous medium system. Various calibrations were performed for accurate conversion of fluorescence intensities to microsphere concentrations. Fluorescence intensities were found to linearly increase with microsphere concentrations (5 x 10(5)-5 x 10(8) spheres/mL in saturated sand) and with camera exposure time. Fluorescence intensities also increased with sand thickness (saturated with microsphere solution), indicating that the fluorescence signals detected by the imaging system were integrated signals from the entire thickness (10 mm) of the sand. A set of microsphere transport experiments was conducted to demonstrate the versatility of the imaging system. Excellent mass recoveries (93-103%) were achieved in all transport experiments, demonstrating the robustness of the imaging system for quantitative study of colloid transport. The system allowed the change of flow velocity, ionic strength, and flow direction within one transport experiment and the real-time, quantitative monitoring of the movement of microspheres in packed sand, greatly reducing the time and effort needed for similar work with traditional column experiments.

Microcracks in compact bone: a three‐dimensional view

Microcracks have been implicated in the loss of bone quality for osteoporosis. In order to detect and monitor their growth, and to use these data to predict fractures, it is essential to obtain quantitative data regarding their shape in three dimensions. Beam-shaped bone samples from sheep radii were prepared and stained with fluorochrome dyes and tested in cyclical fatigue under four-point bending in a servo-hydraulic fatigue-testing machine. Samples were tested at a frequency of 30 Hz under load control at a stress range of 100 MPa. Holes were drilled into bone samples and used as reference points for reconstructions. A series of thin longitudinal sections were cut using a sledge macrotome. A two-dimensional image of each section was examined using an epifluorescence microscope and images transferred to a PC via a CCD low-light colour video camera. A three-dimensional image of each microcrack was reconstructed using computer software, and its dimensions measured. Cracks were elliptical in shape, longer in the longitudinal direction and with a mean aspect ratio of 5.5 +/- 1.05. The mean (+/- SD) length and width of labelled microcracks were 488 +/- 151 and 88 +/- 21 microm, respectively.

Nanoparticle-Assisted Surface Immobilization of Phospholipid Liposomes

Phospholipid liposomes (100-200 nm diameter) are deposited onto solid substrates after stabilizing them against fusion with the solid by allowing charged nanoparticles to adsorb at approximately 25% surface coverage. The immobilized vesicles remain stable over a period of days. Epifluorescence imaging shows that they diffuse freely over surfaces with the same charge but adsorb tightly onto surfaces with opposite charge. Nanoparticle adsorption to surface patterns of opposite charge provides a facile method to create large-scale surface-supported arrays of intact liposomes. This surface attachment method is simple chemically and applies generally for solid surfaces that can be hydrophobic or hydrophilic. Offering routes to localize proteins and other vesicle-contained objects at surfaces in tailored spatial patterns, these immobilized liposome arrays may find diverse applications in the emerging field of nanobiotechnology.

Capture of airborne nanoparticles in swirling flows using non-uniform electrostatic fields for bio-sensor applications

Collection of biological particles is the first and critical step for any biological agent detection system. Towards our goal of capturing and detecting airborne biological entities in real time, here we investigate on the design of an electrostatic particle capture system. We report on the capture of airborne 100 nm diameter polystyrene nanoparticles as a model system, in swirling flows under non-uniform electrostatic fields with an electrospray aerosol generator and a homemade particle collector. The particle collector has five positive electrodes on the bottom and one large grounded electrode on the top. The nanoparticles coming into the collector were slowed down during their swirling and stayed in the collector long before leaving the collector. Silicon chips were placed on the bottom electrodes and the electrostatically captured particles were counted as a function of flow rates, electrode positions, bias voltages, and capture times by epifluorescent images and scanning electron micrographs (SEMs). Particles captured in the electrode at the center of the collector were much less than those on the surrounding four electrodes and 10–25% of the particles with negative charges entering the collector were captured on the bottom electrodes at a flow rate of 1.1 l/min and an applied potential of 2 kV. Particle capture increased with decreasing flow rates. We also simulated flow and electrical fields separately, and found the positional trends to be in good agreement with the measurements. This collector is well adaptable to integration with micro resonator devices and can be used for real-time monitoring of bioaerosols.

PCR-identification of Dunaliella salina (Volvocales, Chlorophyta) and its growth characteristics

The saline pond microalga, Dunaliella salina (Dunal) Teod. maintained in De Walne's (basal) medium under laboratory conditions was confirmed by amplifying the chromosomal DNA of the microalga by PCR with specific primers MA1 and MA2. Seaweed extracts obtained from Sargassum wightii and Ulva lactuca were amended separately at 1.0%, 1.5%, 2.0% and 2.5% levels to the basal medium in order to assess their potential on the growth and concentration of pigments, viz. Chl a, Chl b and beta-carotene of the alga. beta-Carotene was isolated and visible absorption spectrum was taken at 443 and 475 nm confirmed the presence of 9-cis-beta-carotene and all-trans-beta-carotene isomers. Maximum yield, highest division rate (mu) and highest pigment concentrations were observed in the cells grown in 1.5% S. wightii and 2.0% U. lactuca amended medium and these cells were subjected to DAPI staining. The results of epifluorescence microscopy and image analysis revealed a significant enhancement of the cell and nuclear area of the microalgae.

Convective instability of electrokinetic flows in a cross-shaped microchannel

We present a parametric experimental study of convective electrokinetic instability (EKI) in an isotropically etched, cross-shaped microchannel using quantitative epifluorescence imaging. The base state is a three-inlet, one-outlet electrokinetic focusing flow configuration where the centre sample stream and sheath flows have mismatched ionic conductivities. Electrokinetic flows with conductivity gradients become unstable when the electroviscous stretching and folding of conductivity interfaces grows faster than the dissipative effect of molecular diffusion. Scalar images, critical applied fields required for instability, and temporal and spatial scalar energy are presented for flows with a wide range of applied d.c. electric field and centre-tosheath conductivity ratios. These parameters impose variations of the electric Rayleigh number across four orders of magnitude. We introduce a scaling for charge density in the bulk fluid as a function of local maximum conductivity gradients in the flow. This scaling shows that the flow becomes unstable at a critical electric Rayleigh number (Ra e,l =205) and applies to a wide range of applied field and centre-to-sheath conductivity ratios. This work is relevant to on-chip electrokinetic flows with conductivity gradients such as field amplified sample stacking, flow at the intersections of multi-dimensional assays, electrokinetic control and separation of sample streams with poorly specified chemistry, and low-Reynolds number micromixing.

High concordance of drug-induced human hepatotoxicity with in vitro cytotoxicity measured in a novel cell-based model using high content screening

To develop and validate a practical, in vitro, cell-based model to assess human hepatotoxicity potential of drugs, we used the new technology of high content screening (HCS) and a novel combination of critical model features, including (1) use of live, human hepatocytes with drug metabolism capability, (2) preincubation of cells for 3 days with drugs at a range of concentrations up to at least 30 times the efficacious concentration or 100 microM, (3) measurement of multiple parameters that were (4) morphological and biochemical, (5) indicative of prelethal cytotoxic effects, (6) representative of different mechanisms of toxicity, (7) at the single cell level and (8) amenable to rapid throughput. HCS is based on automated epifluorescence microscopy and image analysis of cells in a microtiter plate format. The assay was applied to HepG2 human hepatocytes cultured in 96-well plates and loaded with four fluorescent dyes for: calcium (Fluo-4 AM), mitochondrial membrane potential (TMRM), DNA content (Hoechst 33,342) to determine nuclear area and cell number and plasma membrane permeability (TOTO-3). Assay results were compared with those from 7 conventional, in vitro cytotoxicity assays that were applied to 611 compounds and shown to have low sensitivity (<25%), although high specificity ( approximately 90%) for detection of toxic drugs. For 243 drugs with varying degrees of toxicity, the HCS, sublethal, cytotoxicity assay had a sensitivity of 93% and specificity of 98%. Drugs testing positive that did not cause hepatotoxicity produced other serious, human organ toxicities. For 201 positive assay results, 86% drugs affected cell number, 70% affected nuclear area and mitochondrial membrane potential and 45% affected membrane permeability and 41% intracellular calcium concentration. Cell number was the first parameter affected for 56% of these drugs, nuclear area for 34% and mitochondrial membrane potential for 29% and membrane permeability for 7% and intracellular calcium for 10%. Hormesis occurred for 48% of all drugs with positive response, for 26% of mitochondrial and 34% nuclear area changes and 12% of cell number changes. Pattern of change was dependent on the class of drug and mechanism of toxicity. The ratio of concentrations for in vitro cytotoxicity to maximal efficaciousness in humans was not different across groups (12+/-22). Human toxicity potential was detected with 80% sensitivity and 90% specificity at a concentration of 30x the maximal efficacious concentration or 100 microM when efficaciousness was not considered. We conclude that human hepatotoxicity is highly concordant with in vitro cytotoxicity in this novel model and as detected by HCS.

Behavioral Profiling of Human Transitional Cell Carcinoma Ex vivo

Outcome studies of many types of cancer have revealed that tumors of indistinguishable histologic appearance may differ significantly in aggressiveness and in their response to therapy. A strategy that would enable early identification of patients at high risk for disease progression and allow screening of multiple therapeutic agents simultaneously for efficacy would improve clinical management. We have developed an orthotopic organ culture model of bladder cancer in which quantum dot-based fluorescent imaging approaches are used to obtain quantitative measurements of tumor cell behavior. Human transitional cell carcinoma (TCC) cells are labeled with quantum dot nanoparticles, and the cells instilled into the rat bladder in vivo, after which the bladder is excised and cultured ex vivo. Cell implantation, proliferation, and invasion into the organ wall are monitored using epifluorescence imaging and two-photon laser scanning confocal microscopy. Using this approach, we were able to assign distinct phenotypes to two metastatic bladder cancer cell lines based on different patterns of invasiveness into the bladder wall. We also showed that established tumor cell masses regressed following intravesical administration of the chemotherapeutic drug thiotepa. Collectively, these findings suggest that this assay system, which we have named EViTAS (for ex vivo tumor assay system), can recapitulate salient aspects of tumor growth in the host and is amenable to behavioral profiling of human cancer.

Langmuir and Langmuir–Blodgett films of quantum dots

Trioctylphosphine oxide (TOPO) capped CdSe and (CdSe)ZnS quantum dots (QDs) were prepared. The surface chemistry behavior of both QDs at the air–water interface was carefully examined by various physical measurements. Stable Langmuir films were formed for both QDs. The average limiting nanoparticle area derived from the π– A isotherms of the QDs could be used to estimate the average size of the QDs if the thickness of TOPO shell was counted. The epifluorescence image of the QDs Langmuir films revealed an aggregation in 2D during the early stage of the compression process. On the other hand, a diacetylene-peptide (PDA-Cys-Cys-Gly-OH) derivative, namely PDA-CCG, was used to modify the surface of QDs. This modification resulted in a robust and homogeneous Langmuir film, which is photopolymerizable and contains active binding sites for the binding of biomolecules.

Confocal imaging of porosity in hardened concrete

We present the results of an exploratory 3D study of fine pore structures in hardened cement paste. A laser scanning confocal microscope was used in reflected (epi) fluorescent imaging mode, allowing for the very high optical resolution of features well below 1 μm in size. Images of pore structures were captured using a ‘reverse imaging contrast’ technique where spaces in the material were impregnated with a fluorescent, dyed epoxy-resin, and scanned in the x– y plane by a laser probe. A vertical ‘stack’ of these optical sections was acquired by imaging sequentially through the z-axis. Using this technique it was possible to observe, partially hydrated cement grains, porous natural quartz aggregate interfaces, micro-cracking in the hydrated cement paste and aggregate particles, as well as fine capillary pores and very small air voids. Operating at the limit of its capability the microscope was able to perform high resolution imaging of the internal areas of partially reacted cement grains, and pore structures approximately 0.17 μm across were measured. 3D models were produced to help visualise the true morphology and distribution of porous features.

Electrokinetic Transport in Nanochannels. 2. Experiments

We present an experimental study of nanoscale electrokinetic transport in custom-fabricated quartz nanochannels using quantitative epifluorescence imaging and current monitoring techniques. One aim is to yield insight into electrical double layer physics and study the applicability of continuum theory to nanoscale electrokinetic systems. A second aim is to explore a new separation modality offered by nanoscale electrophoretic separations. We perform parametric variations of applied electric field, channel depth, background buffer concentration, and species valence to impose variations on zeta potential, effective mobility, and Debye length among other parameters. These measurements were used to validate a continuum theory-based analytical model presented in the first of this two-paper series. Our results confirm the usefulness of continuum theory in predicting electrokinetic transport and electrophoretic separations in nanochannels. Our model leverages independent measurements of zeta potential performed in a microchannel system at electrolyte concentrations of interest. These data yield a zeta potential versus concentration relation that is used as a boundary condition for the nanochannel electrokinetic transport model. The data and model comparisons together show that the effective mobility governing electrophoretic transport of charged species in nanochannels depends not only on ion mobility values but also on the shape of the electric double layer and analyte ion valence. We demonstrate a method we term electrokinetic separation by ion valence, whereby both ion valence and mobility may be determined independently from a comparison of micro- and nanoscale transport measurements.

The leading edge is a lipid diffusion barrier

Actin polymerization drives many cellular events, including endocytosis, pathogen rocketing, and cell spreading. Force generation and polymerization regulation are intimately linked where an actin meshwork attaches to, and pushes against, an interface. We reasoned that interaction with actin filament plus-ends might stabilize the position of components within the plasma membrane at the leading edge, thereby slowing the diffusion of lipids within the bilayer where filament growth occurs. To test this hypothesis we focally labeled the outer membrane leaflet of migrating keratocytes and compared the initial diffusion of carbocyanine dyes in the dorsal and ventral lamellipodium membranes using sequential TIRF and epi-fluorescent imaging. Global diffusion analysis shows that lateral mobility of lipids in the outer membrane leaflet is blocked at the leading edge during protrusion. Cytochalasin treatment abolished this diffusion barrier, but we found no evidence to support the involvement of membrane microdomains. Our results demonstrate the immobilization of membrane components at the leading edge, and suggest that interaction between actin filaments and the plasma membrane is mediated by densely packed molecular complexes. We propose that actin polymerization traps regulatory proteins at the leading edge in a positive-feedback loop.

New tools for resolving phylogenies: a systematic revision of the Convolutidae (Acoelomorpha, Acoela)

Molecular sequence data, morphological characters of spermatozoa, and newly obtained morphological characters of penis musculature are used here to revise the systematics of the family Convolutidae (Acoela). Species having isodiametric penes with non-anastomosing longitudinal muscles are transferred to the family Isodiametridae fam. nov. Species with longitudinal penis muscle fibres that anastomose or cross-over each other remain in the Convolutidae. Some species of the genera Convoluta and Conaperta (Convolutidae) are transferred to the genus Isodiametra gen. nov. (Isodiametridae fam. nov.). The genus Stomatricha (Otocelididae) is transferred to the family Convolutidae. Convoluta opisthandropora (Convolutidae) is transferred to the genus Pseudohaplogonaria (Haploposthiidae). Aphanostoma sanguineum (Convolutidae) is transferred to the genus Pseudactinoposthia (Actinoposthiidae). Zusammenfassung Molekulare Sequenzdaten, Spermien-Morphologie und erstmals ermittelte morphologische Merkmale der Penismuskulatur werden herangezogen, um die Verwandtschaftsverhaltnisse innerhalb der Familie Convolutidae (Acoela) neu zu beleuchten. Dabei werden Arten mit isodiametrischen Penes mit nicht anastomosierender Langsmuskulatur in die neue Familie Isodiametridae gestellt, wahrend Arten mit longitudinalen Penis-Muskelfasern, die sich verbunden oder einanderuberkreuzen, innerhalb der Convolutidae belassen. Einige Arten der Gattungen Convoluta und Conaperta (Convolutidae) werden in die neue Gattung Isodiametra gen. nov. (Isodametridae fam. nov.) gestellt. Die Gattung Stomatricha (Otocelididae) wird in die Familie Convolutidae uberfuhrt. Convoluta opisthandropora (Convolutidae) wird in die Gattung Pseudohaplogonaria (Haploposthiidae) gestellt und Aphanostoma sanguineum (Convolutidae) in die Gattung Pseudactinoposthia (Actinoposthiidae).

Deficiency of ATP2C1, a Golgi Ion Pump, Induces Secretory Pathway Defects in Endoplasmic Reticulum (ER)-associated Degradation and Sensitivity to ER Stress

Relatively few clues have been uncovered to elucidate the cell biological role(s) of mammalian ATP2C1 encoding an inwardly directed secretory pathway Ca2+/Mn2+ pump that is ubiquitously expressed. Deficiency of ATP2C1 results in a human disease (Hailey-Hailey), which primarily affects keratinocytes. ATP2C1-encoded protein is detected in the Golgi complex in a calcium-dependent manner. A small interfering RNA causes knockdown of ATP2C1 expression, resulting in defects in both post-translational processing of wild-type thyroglobulin (a secretory glycoprotein) as well as endoplasmic reticulum-associated protein degradation of mutant thyroglobulin, whereas degradation of a nonglycosylated misfolded secretory protein substrate appears unaffected. Knockdown of ATP2C1 is not associated with elevated steady state levels of ER chaperone proteins, nor does it block cellular activation of either the PERK, ATF6, or Ire1/XBP1 portions of the ER stress response. However, deficiency of ATP2C1 renders cells hypersensitive to ER stress. These data point to the important contributions of the Golgi-localized ATP2C1 protein in homeostatic maintenance throughout the secretory pathway.

Correction of Motion Artifact in Cardiac Optical Mapping Using Image Registration

Cardiac motion is one of the main sources of artifacts in epifluorescence imaging experiments. It can cause significant error in electrophysiological measurements such as action potential duration. We present a novel approach that uses image registration based on maximization of mutual information to correct for in-plane cardiac motion in such experiments. The approach is relatively fast (a few seconds per frame) and is performed entirely post acquisition. The image registration approach is an alternative to traditional approaches such as mechanical restraint of the heart or addition of chemical uncouplers. Our results show that the image registration method significantly reduces motion-related artifacts in experimental data.

Fluorene-based fluorescent probes with high two-photon action cross-sections for biological multiphoton imaging applications

Two-photon fluorescence microscopy is a powerful tool for the study of dynamic cellular processes and live-cell imaging. Many commercially available fluorescent probes have been used in multiphoton-based imaging studies despite exhibiting relatively low two-photon absorption cross-section values in the tunability range of ultrafast Ti:sapphire lasers commonly used in multiphoton microscopy imaging. Furthermore, available fluorophores may be plagued with low fluorescence quantum yield and/or photoinstability (i.e., photobleaching) on exposure to the high peak power and photon density provided by the ultrafast laser source. To address the demand for better performing dyes, we prepare fluorophores tailored for multiphoton imaging. These fluorophores are based on the fluorene ring system, known to exhibit high fluorescence quantum yield (>0.7) and high photostability. Furthermore, an amine-reactive fluorescent probe for the covalent attachment onto amine-containing biomolecules is also prepared. Epi-fluorescence and two-photon fluorescence microscopy images of H9c2 rat cardiomyoblasts stained with an efficient two-photon absorbing fluorene fluorophore is demonstrated. Additionally, single-photon spectral characteristics of the amine-reactive fluorophore, as well as the two-photon absorption cross sections of its model adduct in solution, and spectral characterization of a bovine serum albumin (BSA) as a model bioconjugate are presented.

Description of pleural defensive organs in three species of firefly larvae (Coleoptera, Lampyridae)

Although reflex bleeding as a defence mechanism is well characterised in the adult lampyrid little is known of the defensive mechanisms of immature bioluminescent beetles. A detailed comparative examination of the morphology and microstructure of the pleural defensive organs of Lampyris noctiluca L., Luciola cruciata Motschulsky and a Nyctophila species obtained from Amol forest, Iran, using light microscopy and epifluorescent imaging, revealed vesicles continuous with the pleural organ membrane and held in place by a disc structure. Structural differences were apparent between species. We speculate on the function and evolution of these organs among the Lampyridae.