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von Wangenheim D.,Institute of Science and Technology Austria | Hauschild R.,Bioimaging Science and Technology | Friml J.,Institute of Science and Technology Austria
Journal of Visualized Experiments | Year: 2017

One of the key questions in understanding plant development is how single cells behave in a larger context of the tissue. Therefore, it requires the observation of the whole organ with a high spatial- as well as temporal resolution over prolonged periods of time, which may cause photo-toxic effects. This protocol shows a plant sample preparation method for light-sheet microscopy, which is characterized by mounting the plant vertically on the surface of a gel. The plant is mounted in such a way that the roots are submerged in a liquid medium while the leaves remain in the air. In order to ensure photosynthetic activity of the plant, a custom-made lighting system illuminates the leaves. To keep the roots in darkness the water surface is covered with sheets of black plastic foil. This method allows long-term imaging of plant organ development in standardized conditions. © 2017.

Ulyanova A.,Bioimaging Science and Technology | Ulyanova A.,National University of Singapore | To X.V.,Bioimaging Science and Technology | Asad A.B.M.A.,Bioimaging Science and Technology | And 3 more authors.
NeuroImage | Year: 2017

Hypothalamus plays the central role in regulating energy homeostasis. To understand the hypothalamic neurocircuit in responding to leptin, Manganese-Enhanced MRI (MEMRI) was applied. Highly elevated signal could be mapped in major nuclei of the leptin signaling pathway, including the arcuate nucleus (ARC), paraventricular nucleus (PVN), ventromedial hypothalamus (VMH) and dorsomedial hypothalamus (DMH) in fasted mice and the enhancement was reduced by leptin administration. However, whether changes in MEMRI signal reflect Ca2+ channel activity, neuronal activation or connectivity in the leptin signaling pathway are not clear. By blocking L-type Ca2+ channels, the signal enhancement in the ARC, PVN and DMH, but not VMH, was reduced. By disrupting microtubule with colchicine, signal enhancement of the secondary neural areas like DMH and PVN was delayed which is consistent with the known projection density from ARC into these regions. Finally, strong correlation between c-fos expression and MEMRI signal increase rate was observed in the ARC, VMH and DMH. Together, we provide experimental evidence that MEMRI signal could represent activity and connectivity in certain hypothalamic nuclei and hence may be used for mapping activated neuronal pathway in vivo. This understanding would facilitate the application of MEMRI for evaluation of hypothalamic dysfunction in metabolic diseases. © 2016 Elsevier Inc.

Kong K.V.,Bioimaging Science and Technology | Dinish U.S.,Bioimaging Science and Technology | Lau W.K.O.,Singapore General Hospital | Olivo M.,Bioimaging Science and Technology | And 2 more authors.
Biosensors and Bioelectronics | Year: 2014

Conventional nanoparticle based Surface enhanced Raman scattering (SERS) technique for pH sensing often fails due to the aggregation of particles when detecting in acidic medium or biosamples having high ionic strength. Here, We develop SERS based pH sensing using a novel Raman reporter, arene chromium tricarbonyl linked aminothiophenol (Cr(CO)3-ATP), functionalized onto a nano-roughened planar substrates coated with gold. Unlike the SERS spectrum of the ATP molecule that dominates in the 400-1700cm-1 region, which is highly interfered by bio-molecules signals, metal carbonyl-ATP (Cr(CO)3)-ATP) offers the advantage of monitoring the pH dependent strong CO stretching vibrations in the mid-IR (1800-2200cm-1) range. Raman signal of the CO stretching vibrations at ~1820cm-1 has strong dependency on the pH value of the environment, where its peak undergo noticeable shift as the pH of the medium is varied from 3.0 to 9.0. The sensor showed better sensitivity in the acidic range of the pH. We also demonstrate the pH sensing in a urine sample, which has high ionic strength and our data closely correlate to the value obtained from conventional sensor. In future, this study may lead to a sensitive chip based pH sensing platform in bio-fluids for the early diagnosis of diseases.© 2013 Elsevier B.V.

Dinish U.S.,Bioimaging Science and Technology | Balasundaram G.,Bioimaging Science and Technology | Chang Y.-T.,Bioimaging Science and Technology | Chang Y.-T.,National University of Singapore | And 2 more authors.
Scientific Reports | Year: 2014

Surface-enhanced Raman scattering (SERS) technique is becoming highly popular for multiplex biosensing due to the 'fingerprint' Raman spectra from every molecule. As a proof-of-concept, we demonstrated the actively targeted multiplex in vitro and in vivo detection of three intrinsic cancer biomarkers - EGFR, CD44 and TGFβRII in a breast cancer model using three multiplexing capable, biocompatible SERS nanoparticles/nanotags. Intra-tumorally injected antibody conjugated nanotags specifically targeting the three biomarkers exhibited maximum signal at 6â€...hours and no detectable signal at 72â€...hours. However, nanotags without antibodies showed no detectable signal after 6â€...hours. This difference could be due to the specific binding of the bioconjugated nanotags to the receptors on the cell surface. Thus, this study establishes SERS nanotags as an ultrasensitive nanoprobe for the multiplex detection of biomarkers and opens up its potential application in monitoring tumor progression and therapy and development into a theranostic probe.

Kho K.W.,Imperial College London | Kho K.W.,National University of Ireland | Kho K.W.,Bioimaging Science and Technology | Kho K.W.,11 Hospital Drive | And 6 more authors.
ACS Nano | Year: 2012

Figure Persented: We report an observation of a peculiar effect in which the vibrational frequencies of antibody-conjugated SERS-active reporter molecules are shifted in quantitative correlation with the concentration of the targeted antigen. We attribute the frequency shifts to mechanical perturbations in the antibody-reporter complex, as a result of antibody-antigen interaction forces. Our observation thus demonstrates the potentiality of an antibody-conjugated SERS-active reporter complex as a SERS-active nanomechanical sensor for biodetection. Remarkably, our sensing scheme, despite employing only one antibody, was found to be able to achieve detection sensitivity comparable to that of a conventional sandwich immunoassay. Additionally, we have carried out a proof-of-concept study into using multiple "stress-sensitive" SERS reporters for multiplexed detection of antigen-antibody bindings at the subdiffraction limit. The current work could therefore pave the way to realizing a label-free high-density protein nanoarray. © 2012 American Chemical Society.

Perumal J.,Bioimaging Science and Technology | Kong K.V.,Bioimaging Science and Technology | Dinish U.S.,Bioimaging Science and Technology | Bakker R.M.,Data Storage Institute Singapore | And 2 more authors.
RSC Advances | Year: 2014

We report a simple and easy to fabricate random silver film (RSF) as a highly sensitive Surface Enhanced Raman Scattering (SERS) substrate which can be fabricated directly onto a dielectric substrate such as glass. An electron beam evaporation system was used for substrate fabrication. The SERS activity is attributed to the formation of electromagnetic 'hot-spots' on the film. Substrate performance is analyzed by studying the reproducibility and signal enhancement from the Raman active molecule, 2-naphthalene thiol (NT), which is covalently anchored to the substrate. The metal thickness is optimized to achieve the highest SERS enhancement. Based on this study we found that a 7 nm RSF substrate gave the best SERS activity. The SERS signal intensity exhibited by 7 nm RSF is found to be at least 3 orders of magnitude higher than that of a commercial substrate. The SERS enhancement factor is estimated to be ∼1 × 107 with a point-to-point intensity variation of about 12% and it reaches a maximum of 15% for batch-to-batch comparison. The efficacy of this substrate for biosensing is demonstrated by detecting H1 influenza protein, and the detection limit is found to be ∼10 pM when it is used along with a recently established nano-stress SERS sensor, 4-ATP (4-amino-thiophenol), as linker molecule. This detection limit shows a performance superior to conventional ELISA (which has a nM detection limit). These results show promise for the development of a biosensing platform based on the marriage of RSF with nano-stress sensors. This journal is © the Partner Organisations 2014.

Borges J.C.,University of Sao Paulo | Seraphim T.V.,University of Sao Paulo | Seraphim T.V.,University of Campinas | Mokry D.Z.,University of Campinas | And 3 more authors.
PLoS ONE | Year: 2012

Protein folding, refolding and degradation are essential for cellular life and are regulated by protein homeostatic processes such those that involve the molecular chaperone DnaK/Hsp70 and its co-chaperone DnaJ. Hsp70 action is initiated when proteins from the DnaJ family bind an unfolded protein for delivery purposes. In eukaryotes, the DnaJ family can be divided into two main groups, Type I and Type II, represented by yeast cytosolic Ydj1 and Sis1, respectively. Although sharing some unique features both members of the DnaJ family, Ydj1 and Sis1 are structurally and functionally distinct as deemed by previous studies, including the observation that their central domains carry the structural and functional information even in switched chimeras. In this study, we combined several biophysical tools for evaluating the stability of Sis1 and mutants that had the central domains (named Gly/Met rich domain and C-terminal Domain I) deleted or switched to those of Ydj1 to gain insight into the role of these regions in the structure and function of Sis1. The mutants retained some functions similar to full length wild-type Sis1, however they were defective in others. We found that: 1) Sis1 unfolds in at least two steps as follows: folded dimer to partially folded monomer and then to an unfolded monomer. 2) The Gly/Met rich domain had intrinsically disordered characteristics and its deletion had no effect on the conformational stability of the protein. 3) The deletion of the C-terminal Domain I perturbed the stability of the dimer. 4) Exchanging the central domains perturbed the conformational stability of the protein. Altogether, our results suggest the existence of two similar subdomains in the C-terminal domain of DnaJ that could be important for stabilizing each other in order to maintain a folded substrate-binding site as well as the dimeric state of the protein. © 2012 Borges et al.

News Article | February 8, 2017

Imaging very small materials takes not only great skill on the part of the microscopist, but also great instruments and techniques. For a refined microscopic look at biological materials, the challenges include getting an image that is free from "noise," the interference that can be caused by a number of items, including the area surrounding an item. Labels, dyes, or stains that are added in order to see the item more clearly can also present issues as they can affect the item that is to be scanned in unexpected ways--damaging or even killing biological materials. Looking at microtubules is an interesting case in point. The hollow tubular structure serve as a backbone of cells and helps carry materials in the cell. Malfunctioning microtubules have been associated with various illnesses including cancer and Alzheimer's disease. Understanding how microtubules function could be an important step in understanding disease progression. However, studying a single dynamic microtubule, which measures 24 nanometers in diameter, and up to 10 microns in length, is not an easy task. Researchers in the Quantitative Light Imaging Laboratory at the Beckman Institute for Advanced Science and Technology at the University of Illinois have been able to use label-free spatial light interference microscopy (SLIM) and computer processing in order to image the microtubules in an assay. The study, "Label-Free Imaging of Single Microtubule Dynamics Using Spatial Light Interference Microscopy," was recently published in ACS Nano. Being able to see the microtubules without the use of dyes or stains is a major contribution. "The label-free aspect is the main breakthrough in my opinion," said Gabriel Popescu, associate professor of electrical and computer engineering, and member of Beckman's Bioimaging Science and Technology Group. Popescu is the senior author on the study. "There have been other efforts towards making this label-free, it's a very important class of challenges. Current techniques yield smaller fields of view, and the image contrast is not as good." By measuring how much light is delayed through the specimen at all points in the field of view, the researchers are able to find the optical path length map for the sample. This optical path length--or phase information--relates to a sample's refractive index and thickness, enabling detailed studies on cell structure and dynamics. "The instrument provides a blurring of the image that's much bigger than the size of the microtubule," explains Popescu. "So it's as if it's smearing out the values of that phase delay. But since we our system very well, we're able to back it up and come up with an effective index value for the microtubule, which is correct." The numerical processing used provides the sensitivity not only to see the tubules but also is used to measure light scattering. "A key physics point is that once you know both the intensity and phase of the light, then you can numerically process that information and virtually propagate the light anywhere in space, including at a plane far away from the microtubule, in order to study the scattered light," said Popescu. Previous efforts at imaging the miniscule structures have used immunofluorescence, injecting antibodies into fluorescent dyes in order to clearly see the cell as it functions. However, the fluorescence can affect cell function and the length of time that the cell can be imaged. "We imaged them for a very long period of time, not two or three minutes, but more like eight hours," said Mikhail Kandel, a doctoral student in electrical and computer engineering and lead author on the study. "People are interested in the metabolic rates of the proteins that walk on the microtubules and we showed how you can watch the deceleration of these proteins, which is equivalent to monitoring the consumption of their fuel source." "You could potentially figure out the consumption of ATP and motility characteristics of the proteins, which are very interesting." The Beckman researchers worked with Paul Selvin, professor of physics. "This just came out of a discussion with Paul Selvin's group, who have been studying microtubules for a long time using traditional methods of fluorescence," said Popescu. "Mikhail got in contact with his students and they said, let's give it a try. Seeing them with other types of fluorescence is a major improvement because you can basically image them forever." "My group is interested in seeing how proteins move on and around microtubules," said Selvin, one of the study's authors. "This new technique not only enables us to get an idea of how the cells will function over time, but also raises the possibility of in vivo imaging of cells." SLIM is a commercially manufactured product that can fit on to upgrade about any microscope, say the researchers. This allows biologists to use other microscopy techniques, including fluorescence, in addition to SLIM. The SLIM product is available through Phi Optics, a company that Popescu founded. "One of the biggest challenges in interferometry is sensitivity, which is affected drastically by environmental noise, for example, vibrations or air fluctuations. But with the particular stable geometry used in SLIM, we can actually achieve incredible sensitivity in fractions of nanometers," said Popescu. The researchers plan to push the boundaries on imaging cells, hopefully imaging microtubules in live cells. "If we manage to push this in a living cell, that would be a real breakthrough," said Popescu. "We anticipate great challenges because of the background that exists in the cells. Encouraged by these results, we are thinking that one day we might be able to have such a sensitivity to see phase shifts from single molecules. "We're not there yet, but one can dream."

Dos Santos A.E.,Federal University of Rio de Janeiro | Kuster R.M.,Federal University of Rio de Janeiro | Yamamoto K.A.,Federal University of Rio de Janeiro | Salles T.S.,Federal University of Rio de Janeiro | And 5 more authors.
Parasites and Vectors | Year: 2014

Background: The arthropod-borne Mayaro virus (MAYV) causes 'Mayaro fever', a disease of medical significance, primarily affecting individuals in permanent contact with forested areas in tropical South America. Recently, MAYV has attracted attention due to its likely urbanization. Currently, there are no licensed drugs against most mosquito-transmitted viruses. Here, we investigated the in vitro anti-MAYV activity of the flavonoids quercetin and its derivatives from the Brazilian shrub Bauhinia longifolia (Bong.) Steud. Methods. Flavonoids were purified by chromatographic fractionation from leaf extracts of B. longifolia and chemically identified as quercetin and quercetin glycosides using spectroscopic techniques. Cytotoxicity of purified flavonoids and of EtOAc- and n-BuOH-containing flavonoid mixtures was measured by the dye-uptake assay while their antiviral activity was evaluated by a virus yield inhibition assay. Results: The following flavonoids were purified from B. longifolia leaves: non-glycosylated quercetin and its glycosides guaijaverin, quercitrin, isoquercitrin, and hyperin. EtOAc and n-BuOH fractions containing these flavonoids demonstrated the highest antiviral activity of all tested substances, while quercetin had the highest antiviral activity amongst purified flavonoids. Quercetin, EtOAc, or n-BuOH fractions inhibited MAYV production by more than 90% at 25 μg/mL, displaying a stronger antiviral effect than the licensed antiviral ribavirin. A mixture of the isomers isoquercitrin and hyperin had a modest antiviral effect (IC90 = 104.9), while guaijaverin and quercitrin did not show significant antiviral activity. Conclusions: B. longifolia is a good source of flavonoids with anti-Mayaro virus activity. This is the first report of the activity of quercetin and its derivatives against an alphavirus. © 2014 dos Santos et al.; licensee BioMed Central Ltd.

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