Before imaging, culture medium was replaced by Hanks’ balanced salt solution (HBSS) for HeLa cells and cardiac myocytes or Krebs-Ringer-HEPES (KRH) buffer  for PC12 cells. Cells were imaged on an inverted microscope (TE300, Nikon) with a xenon lamp (C6979, Hamamatsu), a 1006 oil immersion objective lens (S Fluor, Nikon), a quad channel imager (Quad-View, Optical Insights), and a cooled CCD camera (CoolSNAP HQ, Roper scientific), and were maintained at 37 uC during all imaging experiments. The quad channel imager mounted three dichroic mirrors: Q500LP; Q530LP; Q570LP, and four emission filters: HQ487/25m; HQ515/30m; HQ550/40m; HQ590/40m. Excitation filters HQ405/206 or D380/106 were used for dualFRET experiments. For comparison of red cameleons, images of directly excited Sapphire and RFP were acquired with a 106 objective lens (Plan Fluor, Nikon) and excitation filters HQ405/206 and S550/206 and emission filters S535/30m and S605/40m without the quad channel imager, respectively. For all excitations, we commonly used a dichroic mirror 455DCLP in the microscope modified as previously described . All filters were obtained from Chroma Technology. Whole system was controlled by using MetaMorph software (Universal Imaging). Timelapse intervals were 50–100 ms and 2.5 s for cardiac myocytes and PC12 cells, respectively, and exposure time was 50–100 ms (464 or 868 binning). Excitationlight is attenuated by a 25% transmittance neutral density (ND) filter to reduce photobleaching, as necessary.
membrane, with regions containing larger amounts of PrP-CFP-GPI and other regions richer in PrP-YFP-GPI. Cell substructure for the double clone was highlighted through nuclei and ER labeling using the ﬂuorophores Hoechst 33342 and ER-tracker red (Molecular Probes), respectively (Fig. 1D, left panel). Non-homogeneous distribution of ﬂuorescent proteins was clearly observed in the three-dimensional view of the cell membrane showing regions where clusters of ﬂuorescent proteins occur (Fig. 1D, right panel). Whether this clustering relates to membrane microdomains such as caveolae or lipid rafts remains to be de- termined, namely because their size seems to be beyond the resolution of light microscopy . Despite recent developments to characterize membrane microdomains, such as photonic force microscopy and single molecule microscopy, controversy still remains and size estimates for these microdomains range from 26 nm to around 700 nm [29,30]. Large microdomains substructure may result from dynamic assemblies of individual rafts [29,31]. Nevertheless, protein clustering on membrane microdomains has been detected usingFRET measurements to near- Angstrom sub-diffraction resolution [19,32,33]. Membrane microdo- mains which form in the exoplasmic lea ﬂet of cellular membranes can selectively incorporate protein and thereby result in protein –protein and protein –lipid interactions . The pattern of ﬂuorescence
was performed on a Zeiss Axiovert 200 inverted microscope at the University of Wuerzburg (Wuerzburg, Germany), equipped with an oil immersion 63x objective lens and a dual-emis- sion photometric system (Till Photonics) as described before . The transfected cells were excited withlight from a polychrome IV (Till Photonics). Illumination was set to 40ms out of a total integration time of 100ms. CFP (480 ± 20 nm), YFP (535 ± 15 nm), and FRET ratio (YFP/ CFP) signals were recorded simultaneously (beam splitter DCLP 505 nm) upon excitation at 436 ± 10 nm (beam splitter DCLP 460 nm). Fluorescence signals were detected by photodiodes and digitalized using an analogue-digital converter (Digidata 1440A, Axon Instruments). All data were recorded on a PC running Clampex 10.3 software (Axon Instruments). Resulting individual traces were fit to a one component exponential decay function to extract the expo- nential time constant, tau . The halftime of activation (t 1/2 ) is defined as τ ln2. In dynamic
In summary, we have shown that of the four markers proposed, only two are electrochemically active (methyl nicotinate and 2-methoxybiphenyl). These two markers have been shown to be readily determined using cost-effective, disposable and single-shot screen-printed sensors; ideal for point-of-care and clinical devices. Furthermore, the determination of the markers has been expanded to include their monitoring simultaneously within the same solution which deduced that both can be monitored down to mid-level micromolar concentrations with relative ease.
1.5610 4 MB231_CFP-YFP cells were cultured on 4 well chambered borosilicate coverglass systems (Nunc, Rochester, NY) for one day before the experiment. Cells were treated with various concentrations of TRAIL (0–25 ng/ml) for 2.5 h before imaging. The cells were imaged with a 63x objective lens and a Zeiss Cell Observer Spinning Disk Confocal Microscope system (Thornwood, NY) with a PECON environmental chamber. FRET efficiency was determined by detecting sensitized emission [22,23]. Excitation laser lines with a wavelength of 458, 514, and 458 nm were used for donor CFP, acceptor YFP, and FRET channels, respectively. The emission filter for CFP was 485/30 while the emission filter for YFP and FRET was 535/30. Dichroic beam splitter for Yokogawa confocal scan head was RIFT 457/514/ 647 nm. After obtaining the correction factors for singly expressed CFP and YFP, cell images were acquired for MB231_CFP-YFP cells via CFP, YFP, and FRET channels in an identical manner. Data sets of 9 images were stored as zvi format for future quantitative analysis. Zeiss Axiovision software (ver. 4.8.2) was used for sensitized emission FRET measurement. The FRET efficiency was calculated based on a three-filter system, which is normalized against donor and acceptor intensities, allowing calculation of cross talk and verification of true FRET signal [24,25]. The value of FRET efficiency was calculated according to Xia’s formula . Three randomly selected images were obtained for each experiment. From these images the FRET efficiency was calculated. Two to three regions of interest (ROI) were placed for a respective cell and ten to fifteen ROIs were subjected to FRET analysis.
Full-length P. falciparum P2 protein (PfP2), and certain carboxy- terminal deletion constructs, (P2Cdel20 and P2Cdel40), were expressed as His-tag fusion proteins, and several monoclonal antibodies (mAbs) were generated against these (Figure 1A, B). Full length P. falciparum P1 protein (PfP1) was expressed as a GST- fusion protein. GST-PfP0C, which contained 80% of C-terminal PfP0 protein , and the PfP0-specific mAb E5F4  were also used in this study. Amongst the panel of monoclonals generated against the PfP2 protein, two classes of mAbs were detected; the E2G12 type and the A12D9 type (Figure 1BI). The A12D9 type mapped against the carboxy-terminal domain and cross-reacted with PfP1; while E2G12 set did not react with PfP1 (Figure 1BI). The polyclonal antibody raised against PfP1 did not show cross- reactivity against PfP2 under native condition (Figure 1BII). Anti- P2 mAb E2G12 was specific for Plasmodium P2 protein and reacted specifically with the 27–49 amino-acids (aa) peptide (Figure 1BIII) while A12D9 mapped to the 92–112 amino acids extreme-C- terminal region of P2 (Figure 1BI, Cc). All recombinant PfP2 proteins, the full length as well as the carboxy-terminal deletions, exhibited distinct higher oligomers that were SDS-resistant, while the recombinant PfP1 or PfP0 proteins did not exhibit any oligomerization (Figure 1C). In the crude Plasmodium protein extracts, resolved on SDS-PAGE, a protein band at about 65 kDa was noted in addition to the 16 kDa PfP2 monomer (Figure 1DI). The mAb E2G12 was found to be specific for Plasmodium P2 protein, and did not react with either recombinant PfP1 or the P1 protein in the parasite extract (Figure 1 B, C, D). Although the rabbit polyclonal anti-PfP1 antibody reacted with both PfP1 and PfP2 in denatured conditions (Figure 1DII), it did not react to recombinant PfP2 under native conditions (Figure 1BII). The anti- PfP1 antibodies also showed features distinct from anti-PfP2 in sub-cellular localizations
Cy7 (gray) pairs were calculated as 5.4-nm, 6.2-nm, and 3.8-nm, respectively. (c) A schematic diagram of ALEX three-color confocal setup. The setup was built based on an inverted microscope (TE2000-U, Nikon, Tokyo, Japan) equipped with a three dimensional piezo-stage (LP-100, MadCityLabs, Madison, WI). Two excitation lasers, a diode-pump solid state laser (532-nm, Excelsior-CDRH, Spectra-Physics, Santa Clara, CA) and HeNe laser (633- nm, HRP050, Thorlabs, Newton, NJ), were alternatively switched on and off by using electro-optic modulators (EOM, 350-50, Conoptics, Danbury, CT). To make sure that the two excitation lasers excite the same molecule, they were coupled into a single-mode fiber (460HP, Thorlabs). An oil-immersion objective (UPLSAPO 1006, Olympus, Tokyo, Japan) was used for both the excitation of molecules and the collection of fluorescence signals. The fluorescence signals are measured by using avalanche photo diodes (APD, SPCM-AQRH-14, Perkin Elmer, Wellesley, MA). The identities of other optics are: D1, a dichroic mirror (z532bcm, Chroma, Rockingham, VT); D2, dichroic mirror (z532/633rpc, Chroma); P, pinhole (P75S, Thorlabs); L1 and L2, lens (LAO-90.0-25.0/078, CVI, Irvine, CA); D3, dichroic mirror (640dcxr, Chroma); D4, dichroic mirror (740dcxr, Chroma); F1, bandpass filter (HQ580/60m-2p, Chroma); F2, bandpass filter (HQ680/60m-2p, Chroma); F3, bandpass filter (HQ790/80m, Chroma).
The screening of large numbers of compounds or siRNAs is a mainstay of both academic and pharmaceutical research. Most screens test those interventions against a single biochemical or cellular output whereas recording multiple complementary outputs may be more biologically relevant. High throughput, multi-channel fluorescence microscopy permits multiple outputs to be quantified in specific cellular subcompartments. However, the number of distinct fluorescent outputs available remains limited. Here, we describe a cellular bar-code technology in which multiple cell-based assays are combined in one well after which each assay is distinguished by fluorescence microscopy. The technology uses the unique fluorescent properties of assay-specific markers comprised of distinct combinations of different ‘red’ fluorescent proteins sandwiched around a nuclear localization signal. The bar-code markers are excited by a common wavelength of light but distinguished ratiometrically by their differing relative fluorescence in two emission channels. Targeting the bar-code to cell nuclei enables individual cells expressing distinguishable markers to be readily separated by standard image analysis programs. We validated the method by showing that the unique responses of different cell-based assays to specific drugs are retained when three assays are co-plated and separated by the bar-code. Based upon those studies, we discuss a roadmap in which even more assays may be combined in a well. The ability to analyze multiple assays simultaneously will enable screens that better identify, characterize and distinguish hits according to multiple biologically or clinically relevant criteria. These capabilities also enable the re-creation of complex mixtures of cell types that is emerging as a central area of interest in many fields.
Secreted cysteine-rich Wnt molecules constitute a highly conserved family of growth factors which consists of 21 genes in vertebrates (see wnt homepage at: http://www.stanford.edu/ group/nusselab/cgi-bin/wnt/). Wnt proteins activate different signaling cascades, including the Wnt/b-catenin, Wnt-Calcium and Wnt planar cell polarity pathways. These Wnt triggered pathways interact on several levels of signal transduction to specify the cellular response to any given ligand and/or ligand combina- tion. Thus, they should rather be considered as a Wnt-signaling network [1,2]. Common to all Wnt pathways is the binding of a ligand to seven-pass transmembrane receptors of the frizzled (Fz) family and the regulation of the intracellular adapter protein dishevelled (dsh). The x-ray structure of the Xwnt8/Fz-CRD complex revealed that Wnts interact with the cysteine-rich extracellular domain (CRD) of Fz via two hydrophobic interaction sites . Importantly, the interaction sites of the Wnt ligand, the fatty acid modification and the cysteine-rich C-terminus are highly conserved among all Wnt proteins, including those activating non- canonical pathways. The decision which of the Wnt pathways is activated depends not only on the Wnt/Fz interaction but also on
Boundary conditions. The boundary condition on the OP surfaces (Figure S2 in File S1) was set to opening, with 100,000 Pa average absolute pressure, which allows both in- and outflow without specifying the direction of the flow through the surface. The side walls were set to symmetry, which does not allow the formation of radial velocity components. Both the surface of the cell under the micropipette and the wall of the micropipette were modelled by no-slip boundary condition (NSW in Figure S2 in File S1). The upper end of the pipette (where the fluid leaves the domain) was set to prescribed flow rate, allowing the formation of the outlet velocity profile. The bottom of the Petri dish required special attention when defining the boundary conditions. The majority of the pressure drop (friction losses) is generated on this surface and it is known that the classic no-slip wall boundary condition does not necessarily hold in the case of micro-scale fluid mechanical applications with partially hydrophobic surfaces [37– 43]. However, determining the so-called ‘‘slipping depth’’ – the virtual depth where the velocity profile reaches the zero value - is rather cumbersome as one needs to measure the velocity profile in the vicinity of the wall. Moreover, the wall treatment of ANSYS CFX allows (besides the standard free-slip and no-slip boundary condition) only to specify the shear stress at the wall, i.e. the slope of the wall-parallel velocity component in the wall-normal direction. Hence we decided to perform each computation twice: with no-slip and free-slip settings at the bottom of the Petri dish and compare the results with experimental measurements of the flow rate. The real-life velocity profile (and other integral quantities such as the pressure drop) is expected to lie between these two extreme cases. As the results of simulations with the free- slip condition on the bottom of the Petri dish was very close to the experimental calibration curve, we calculated the lifting force acting on the cell from these simulations.
In this limited cohort of studies, the main results can be summarized as follows: 1). When assessed base on coronary territory, the diagnostic performance of perfusion-CMR assessed as SEN, SPE, and the area under the ROC curve (AUC) were superior over SPECT in detecting CAD. This findings of our study were in accord with the meta-analysis of Iwata et al. , which only included studies assessed base on coronary territory. 2). When assessed base on patient, the diagnostic performance of perfusion- CMR assessed as SEN and AUC values were superior over SPECT in detecting CAD, but inferior in SPE. Two previous indirect comparative meta-analyses demonstrated significantly higher SEN and DOR than SPECT but failed to show significant
Our STED nanoscope setup  featured a 532 nm (PicoTA, PicoQuant, Berlin, Germany) and a 635 nm (LDH-D-C-635, PicoQuant) pulsed diode laser for excitation and a Ti:Sapphire laser (Mira900, Coherent, Santa Clara, CA) for STED, which was tuned to 740 or 760 nm and operating either in the CW or in the mode-locked pulsed mode with a repetition rate of 76 MHz. The STED light was guided through two glass rods and coupled into a 120 m long polarization maintaining single mode fiber (AMS Technology, Mu¨nchen, Germany), which in the pulsed modality stretched the pulse width to ,250 ps. In the pulsed STED modality, the excitation diode lasers were synchronized to the STED laser by a home-built electronic delay unit. In the CW modality, the repetition of the pulsed excitation lasers was tuned to 40 or 80 MHz, based on the application. The doughnut-like intensity distribution of the STED light was created by introducing a polymeric phase plate (RPC Photonics, Rochester, NY) applying a helical phase ramp of exp(iQ), with 0,Q,2p in the STED beam that was then imaged into the back aperture of a 1.4 NA objective lens (HCX PL APO, 1006/1.40, oil, Leica, Wetzlar, Germany). Excitation and STED beams were aligned on the same optical axis using custom-made dichroic mirrors (AHF Analysentechnik, Tu¨bingen, Germany). The fluorescence was detected through the same objective lens, filtered out with appropriate bandpass filters to reject laser scattering and imaged onto a multimode optical fibre with an opening of the size of about an Airy disc of the imaged excitation PSF. The fibre was attached to a single-photon- counting module (id100-MMF50, id Quantique, Carouge, Swit- zerland) and connected to a time-correlated single-photon- counting board (SPC-730, Becker & Hickl GmbH, Berlin, Germany). The image acquisition was performed by scanning the sample with a 3D piezo stage (NanoMax TS 3-axis, Thorlabs GmbH Europe, Dachau, Germany). The STED and confocal reference images were recorded simultaneously on a line-by-line basis by opening and closing a mechanical shutter in the STED beam.
Remarkably, RNA release was found to occur almost immediately following internalization of the virus particles extremely close to the cell surface, in vesicles that were located within 100–200 nm of the plasma membrane (Figure 2C). Whereas the site of RNA release was more precisely determined for the adhering surface of the cell by TIRF, we showed, using confocal microscopy, that RNA release took place close to either the top (apical) or the bottom (attached) surface of cells with similar kinetics (Figure 2A). Inhibitors that disrupt actin cytoskeleton, deplete ATP, or inhibit tyrosine kinases totally block infection from both surfaces (Figures 5–8). Thus, the entry pathways from the two surfaces of nonpolarized cells are most likely similar, if not identical. The demonstration that genome release takes place from vesicles very near the cell surface is consistent with previous observations that endocytic acidiﬁcation and intact micro- tubules are not required for PV infection [32,33]. Indeed, we have conﬁrmed that neither raising endocytic pH nor disrupting microtubules inhibits either RNA release or infection (Figure 6). Interestingly, shortly after RNA release, the empty capsids were observed to move rapidly along microtubules. However, this transport behavior is unlikely to be relevant to PV infection. Our results challenge previous thoughts that PV RNA release may occur at the plasma membrane or near the nucleus . They also raise the question of what triggers RNA release from the vesicles near the plasma membrane. One potential candidate arises from the curvature of the vesicles enveloping the viral particles, which may allow additional copies of the N-terminus of VP1 to be inserted into the vesicle membrane or allow more intimate association of the virus particles with the membrane, generating mechanical forces that trigger RNA release. It is also possible that internalization results in the recruitment of cellular factors that facilitate RNA release and
materials, calculated from Ref. . Although the general trend is followed by both sets of points, a systematic difference is observed as the measured efﬁciency is always 10–20% lower than the theoretical one. This discrepancy is more signiﬁ- cant for lower energies, implying that it may be due to absorptions in the detector cover (made of aluminium foil and black tape, used to block ambient light). Other contributions may come from a detector active thickness smaller than 300 mm (though the detector operated above full depletion, the actual thickness ofits active area was never directly measured) or a normalization error due to the (3-year old) absolute calibration of the CZT reference detector.
All MRI experiments were conducted on a 9.4T horizontal animal magnet (Magnex Scientific, Abingdon, UK) interfaced to a Varian INOVA console (Varian, Palo Alto, CA, USA). A butter- fly-shape 1 H surface coil (2.8×2.0 cm with the short axis paralleled to the animal spine) was used to collect all MRI data. Scout images were acquired using a turbo fast low angle shot (Tur- boFLASH) imaging sequence  with the following acquisition parameters: TR = 10 ms, TE = 4 ms, image slice thickness = 2 mm, field of view (FOV) = 3.2 cm×3.2 cm; image matrix size = 128×128.
An inverted microscope (IX-71; Olympus) equipped with a 100 W halogen lamp and a condenser (numerical aperture, NA = 0.5) or an upright microscope (BX53; Olympus) equipped with a 100 W halogen lamp and a condenser (NA = 0.9) were used for imaging. To obtain the highest light intensity, all factors affecting the brightness of the halogen lamp needed to be removed from the optical path before observation. Usually, there is some filter between the condenser and halogen lamp, and these filters greatly reduce the intensity of halogen light. If the condenser itself also includes a filter, such as that found on differential interference contrast (DIC) microscopes, it should also be removed. The diaphragm of the halogen lamp should be open completely. We developed the new filter adapter withexcitation filter and diaphragm with support from Olympus, Tokyo, Japan. This filter adapter was a prototype and is not for sale at present, but Olympus plans to make it available commercially soon. To observe GFP and Alexa Fluor 488, a 460–490 nm bandpass (BP) filter was used for excitation and a 510 nm barrier (BA) filter was used to collect fluorescent light. To observe phycoerythrin, Cy3, Alexa Fluor 555 and Alexa Fluor 546, a 480–555 BP filter and 580 nm BA filter were used. BA filters were placed in filter cubes or in the eyepiece. The excitation filter for the mercury vapor lamp and a dichroic mirror were removed from the cube. Because of the strong emission of the phycoerythrin dye, the type of objective lens used for its observation is not important. In this study, an Olympus LCPlanFl 0.4 NA objective lens (620) and plastic dishes were used for fluorescence observation or enucleation. For observing other dyes, an Olympus UPlanSApo 0.75 NA objective lens (620) and a glass-bottomed dish were needed.
Our analyses suggest that data from experimental FRET pairs should be compared carefully to data from all appropriate negative control FRET pairs in order to determine whether FRET values are significant. Furthermore, the required correction methods and controls also depend on the relative expression levels of the FRET donor and acceptor. Finally, using more than one method to determine FRET values for the same cell provides independent verification of the data. This comprehensive confocal microscopy approach to FRET analysis may be broadly useful for the characterization of direct protein-protein interactions in fixed cells. In addition to their ability to undergo BAR domain-mediated dimerization and membrane targeting, APPL proteins exhibit PH and PTB domain-mediated phosphoinositide binding [19,20] and membrane targeting . Dynamic associations between APPL proteins and cell membranes are likely to be coordinately regulated by BAR domain-mediated dimerization, phosphoinosi- tide binding, and interactions with protein binding partners, including transmembrane receptors, signaling proteins, and GTP- bound RAB5. The APPL1 BAR and PH domains are required for interaction with GTP-RAB5 . Analysis of the APPL1 BAR- PH domain crystal structure together with in vitro binding studies suggests that APPL1 BAR-PH homodimers form heterotypic RAB5 binding platforms in which the BAR domain of one monomer and the PH domain of a second monomer interact with GTP-RAB5 on each end of the curved BAR-PH dimer . Although GTP-RAB5 interacts with both APPL1 and APPL2 , direct interaction between GTP-RAB5 and APPL1 homo- dimers, APPL2 homodimers, or APPL1-APPL2 heterodimers on cell membranes has not been demonstrated. However, overex- pression of APPL1-YFP or APPL2-YFP leads to the recruitment of endogenous RAB5 to enlarged APPL-associated cytosolic mem- brane structures .
Destruxins (Dtx) are cyclodepsipeptides produced by enthomopathogenic fungi, which are used in biological control of different agricultural insect plagues. The present investigation reports a new approach for analysis of destruxins produced by the fungal strain Beauveria felina, using LC-PDA-ELSD-MS. Compared to previous methods, the new approach uses a clean-up on C 18 cartridges, which effectively removes growth media constituents. Moreover, the use of 50:50 (v/v) MeCN/MeOH as the strongest eluting solvent in a gradient system over 0.1% H 2 O proved to give a better resolution of chromatographic peaks. Simultaneous detection using photodiode array (PDA), evaporative light scattering detector (ELSD) and mass spectrometry (MS) indicated a practically identical response of all detectors for destruxins analysis. Five samples obtained from the culture media of B. felina were analysed, and indicated the presence of twenty known destruxins and six yet unreported cyclodepsipeptides. Considering the reduced use of MeCN, and the effectiveness of ELSD as a detector for destruxins, the method proved to be valuable and cost-effective for quality control analysis of destruxin-producing fungal strains.
OBJECTIVE: To assess the role of proton magnetic resonance spectroscopy and dynamic contrast-enhanced magnetic resonance imaging in the differentiation between malignant and benign musculoskeletal tumors. MATERIALS AND METHODS: Fifty-five patients with musculoskeletal tumors (27 malignant and 28 benign) were studied. The examinations were performed in a 1.5 T magnetic resonance scanner with standard protocol, and single voxel proton magnetic resonance spectroscopy with 135 msec echo time. The dynamic contrast study was performed using T1-weighted gradient-echo sequence after intravenous gadolinium injection. Time- signal intensity curves and slope values were calculated. The statistical analysis was performed with the Levene’s test, followed by a Student’s t-test, besides the Pearson’s chi-squared and Fischer’s exact tests. RESULTS: Proton magnetic resonance spectroscopy sensitivity, specificity and accuracy were, respectively, 87.5%, 92.3% and 90.9% (p < 0.0001). Statistically significant difference was observed in the slope (%/min) between benign (mean, 27.5%/min) and malignant (mean, 110.9%/min) lesions (p < 0.0001). CONCLUSION: The time-intensity curve and slope values using dynamic-enhanced perfusion magnetic resonance imaging in association with the presence of choline peak demonstrated by single voxel magnetic resonance spectroscopy study are useful in the differentiation between malignant and benign musculoskeletal tumors.
According to the two-way analysis of variance, only the independent factor “adhesive” (F 1, 24 = 29,24, p=0.0001) and the “interaction factor” between the adhesive system and the composite (F 1, 24 = 25,29, p=0.0001) were statistically significant. Tukey’s test (p<0.05) (Table 2) showed that there was no statistically significant difference (p>0.05) between the adhesives when they were combined to the light- activated composite. When in combination with the chemically activated resin, One Step adhesive system had higher bond strengths than Prime & Bond NT.