Red and Green Fluorescence from Oral Biofilms.

PubMed

Volgenant, Catherine M C; Hoogenkamp, Michel A; Krom, Bastiaan P; Janus, Marleen M; Ten Cate, Jacob M; de Soet, Johannes J; Crielaard, Wim; van der Veen, Monique H

2016-01-01

Red and green autofluorescence have been observed from dental plaque after excitation by blue light. It has been suggested that this red fluorescence is related to caries and the cariogenic potential of dental plaque. Recently, it was suggested that red fluorescence may be related to gingivitis. Little is known about green fluorescence from biofilms. Therefore, we assessed the dynamics of red and green fluorescence in real-time during biofilm formation. In addition, the fluorescence patterns of biofilm formed from saliva of eight different donors are described under simulated gingivitis and caries conditions. Biofilm formation was analysed for 12 hours under flow conditions in a microfluidic BioFlux flow system with high performance microscopy using a camera to allow live cell imaging. For fluorescence images dedicated excitation and emission filters were used. Both green and red fluorescence were linearly related with the total biomass of the biofilms. All biofilms displayed to some extent green and red fluorescence, with higher red and green fluorescence intensities from biofilms grown in the presence of serum (gingivitis simulation) as compared to the sucrose grown biofilms (cariogenic simulation). Remarkably, cocci with long chain lengths, presumably streptococci, were observed in the biofilms. Green and red fluorescence were not found homogeneously distributed within the biofilms: highly fluorescent spots (both green and red) were visible throughout the biomass. An increase in red fluorescence from the in vitro biofilms appeared to be related to the clinical inflammatory response of the respective saliva donors, which was previously assessed during an in vivo period of performing no-oral hygiene. The BioFlux model proved to be a reliable model to assess biofilm fluorescence. With this model, a prediction can be made whether a patient will be prone to the development of gingivitis or caries.

Red fluorescent biofilm: the thick, the old, and the cariogenic

PubMed Central

Volgenant, Catherine M.C.; Hoogenkamp, Michel A.; Buijs, Mark J.; Zaura, Egija; ten Cate, Jacob (Bob) M.; van der Veen, Monique H.

2016-01-01

Background Some dental plaque fluoresces red. The factors involved in this fluorescence are yet unknown. Objective The aim of this study was to assess systematically the effect of age, thickness, and cariogenicity on the extent of red fluorescence produced by in vitro microcosm biofilms. Design The effects of biofilm age and thickness on red fluorescence were tested in a constant depth film fermentor (CDFF) by growing biofilms of variable thicknesses that received a constant supply of defined mucin medium (DMM) and eight pulses of sucrose/day. The influence of cariogenicity on red fluorescence was tested by growing biofilm on dentin disks receiving DMM, supplemented with three or eight pulses of sucrose/day. The biofilms were analyzed at different time points after inoculation, up to 24 days. Emission spectra were measured using a fluorescence spectrophotometer (λexc405 nm) and the biofilms were photographed with a fluorescence camera. The composition of the biofilms was assessed using 454-pyrosequecing of the 16S rDNA gene. Results From day 7 onward, the biofilms emitted increasing intensities of red fluorescence as evidenced by the combined red fluorescence peaks. The red fluorescence intensity correlated with biofilm thickness but not in a linear way. Biofilm fluorescence also correlated with the imposed cariogenicity, evidenced by the induced dentin mineral loss. Increasing the biofilm age or increasing the sucrose pulsing frequency led to a shift in the microbial composition. These shifts in composition were accompanied by an increase in red fluorescence. Conclusions The current study shows that a thicker, older, or more cariogenic biofilm results in a higher intensity of red fluorescence. PMID:27060056

Identifying the Assembly Configuration and Fluorescence Spectra of Nanoscale Zinc-Tetraphenylporphyrin Aggregates with Scanning Tunneling Microscopy

PubMed Central

Zhang, Xiao-Lei; Jiang, Jian-Wei; Liu, Yi-Ting; Lou, Shi-Tao; Gao, Chun-Lei; Jin, Qing-Yuan

2016-01-01

ZnTPP (Zinc-Tetraphenylporphyrin) is one of the most common nanostructured materials, having high stability and excellent optoelectronic properties. In this paper, the fluorescence features of self-assembled ZnTPP monomers and aggregates on Au(111) surface are investigated in detail on the nanometer scale with scanning tunneling microscopy (STM). The formation of ZnTPP dimers is found in thick layers of a layer-by-layer molecular assembly on Au substrate with its specific molecular arrangement well characterized. Tip-induced luminescence shows a red shift from tilted dimers comparing with the behavior from monomers, which can be attributed to the change of vibrational states due to the intermolecular interaction and the increasing dielectric effect. The nanoscale configuration dependence of electroluminescence is demonstrated to provide a powerful tool aiding the design of functional molecular photoelectric devices. PMID:26948654

Adsorption properties of BSA and DsRed proteins deposited on thin SiO2 layers: optically non-absorbing versus absorbing proteins

NASA Astrophysics Data System (ADS)

Scarangella, A.; Soumbo, M.; Villeneuve-Faure, C.; Mlayah, A.; Bonafos, C.; Monje, M.-C.; Roques, C.; Makasheva, K.

2018-03-01

Protein adsorption on solid surfaces is of interest for many industrial and biomedical applications, where it represents the conditioning step for micro-organism adhesion and biofilm formation. To understand the driving forces of such an interaction we focus in this paper on the investigation of the adsorption of bovine serum albumin (BSA) (optically non-absorbing, model protein) and DsRed (optically absorbing, naturally fluorescent protein) on silica surfaces. Specifically, we propose synthesis of thin protein layers by means of dip coating of the dielectric surface in protein solutions with different concentrations (0.01-5.0 g l-1). We employed spectroscopic ellipsometry as the most suitable and non-destructive technique for evaluation of the protein layers’ thickness and optical properties (refractive index and extinction coefficient) after dehydration, using two different optical models, Cauchy for BSA and Lorentz for DsRed. We demonstrate that the thickness, the optical properties and the wettability of the thin protein layers can be finely controlled by proper tuning of the protein concentration in the solution. These results are correlated with the thin layer morphology, investigated by AFM, FTIR and PL analyses. It is shown that the proteins do not undergo denaturation after dehydration on the silica surface. The proteins arrange themselves in a lace-like network for BSA and in a rod-like structure for DsRed to form mono- and multi-layers, due to different mechanisms driving the organization stage.

Interconversion of Anthozoa GFP-like fluorescent and non-fluorescent proteins by mutagenesis

PubMed Central

Bulina, Maria E; Chudakov, Dmitry M; Mudrik, Nikolay N; Lukyanov, Konstantin A

2002-01-01

Background Within the family of green fluorescent protein (GFP) homologs, one can mark two main groups, specifically, fluorescent proteins (FPs) and non-fluorescent or chromoproteins (CPs). Structural background of differences between FPs and CPs are poorly understood to date. Results Here, we applied site-directed and random mutagenesis in order to to transform CP into FP and vice versa. A purple chromoprotein asCP (asFP595) from Anemonia sulcata and a red fluorescent protein DsRed from Discosoma sp. were selected as representatives of CPs and FPs, respectively. For asCP, some substitutions at positions 148 and 165 (numbering in accordance to GFP) were found to dramatically increase quantum yield of red fluorescence. For DsRed, substitutions at positions 148, 165, 167, and 203 significantly decreased fluorescence intensity, so that the spectral characteristics of these mutants became more close to those of CPs. Finally, a practically non-fluorescent mutant DsRed-NF was generated. This mutant carried four amino acid substitutions, specifically, S148C, I165N, K167M, and S203A. DsRed-NF possessed a high extinction coefficient and an extremely low quantum yield (< 0.001). These spectral characteristics allow one to regard DsRed-NF as a true chromoprotein. Conclusions We located a novel point in asCP sequence (position 165) mutations at which can result in red fluorescence appearance. Probably, this finding could be applied onto other CPs to generate red and far-red fluorescent mutants. A possibility to transform an FP into CP was demonstrated. Key role of residues adjacent to chromophore's phenolic ring in fluorescent/non-fluorescent states determination was revealed. PMID:11972899

Efficient triplet harvesting of hybrid white organic light-emitting diodes using thermally activated delayed fluorescence green emitter

NASA Astrophysics Data System (ADS)

Lee, Song Eun; Lee, Ho Won; Baek, Hyun Jung; Yun, Tae Jun; Yun, Geum Jae; Kim, Woo Young; Kim, Young Kwan

2016-10-01

Hybrid white organic light-emitting diodes (WOLEDs) were fabricated by applying triplet harvesting (TH) using a green thermally activated delayed fluorescence (TADF) emitter. The triplet exciton of the green TADF emitter can be upconverted to its singlet state. The TH involved energy transfer of triplet exciton from a blue fluorescent emitter to a green TADF and red phosphorescent emitters, where they can decay radiatively. In addition, the triplet exciton of the green TADF emitter was energy transferred to its singlet state for a reverse intersystem crossing by green emission. Enhanced hybrid WOLEDs were demonstrated using an efficient green TADF emitter combined with red phosphorescent and blue fluorescent emitters. Hybrid WOLEDs were fabricated with various hole-electron recombination zones as changing blue emitting layer thicknesses. Among these, hybrid WOLEDs showed a maximum external quantum efficiency of 11.23%, luminous efficiency of 29.20 cd/A, and a power efficiency of 26.21 lm/W. Moreover, the WOLED exhibited electroluminescence spectra with Commission International de L'Éclairage chromaticity of (0.38, 0.36) at 1000 cd/m2 and a color rendering index of 82 at a practical brightness of 20,000 cd/m2.

Photoconversion in orange and red fluorescent proteins

PubMed Central

Kremers, Gert-Jan; Hazelwood, Kristin L.; Murphy, Christopher S.; Davidson, Michael W.; Piston, David W.

2009-01-01

We report that photoconversion is fairly common among orange and red fluorescent proteins, as a screen of 12 variants yielded 8 that exhibit photoconversion. Specifically, three red fluorescent proteins can be switched into a green state, and two orange variants can be photoconverted to the far red. The orange highlighters are ideal for dual-probe highlighter applications, and they exhibit the most red-shifted excitation of all fluorescent protein described to date. PMID:19363494

Red fluorescence in reef fish: A novel signalling mechanism?

PubMed Central

Michiels, Nico K; Anthes, Nils; Hart, Nathan S; Herler, Jürgen; Meixner, Alfred J; Schleifenbaum, Frank; Schulte, Gregor; Siebeck, Ulrike E; Sprenger, Dennis; Wucherer, Matthias F

2008-01-01

Background At depths below 10 m, reefs are dominated by blue-green light because seawater selectively absorbs the longer, 'red' wavelengths beyond 600 nm from the downwelling sunlight. Consequently, the visual pigments of many reef fish are matched to shorter wavelengths, which are transmitted better by water. Combining the typically poor long-wavelength sensitivity of fish eyes with the presumed lack of ambient red light, red light is currently considered irrelevant for reef fish. However, previous studies ignore the fact that several marine organisms, including deep sea fish, produce their own red luminescence and are capable of seeing it. Results We here report that at least 32 reef fishes from 16 genera and 5 families show pronounced red fluorescence under natural, daytime conditions at depths where downwelling red light is virtually absent. Fluorescence was confirmed by extensive spectrometry in the laboratory. In most cases peak emission was around 600 nm and fluorescence was associated with guanine crystals, which thus far were known for their light reflecting properties only. Our data indicate that red fluorescence may function in a context of intraspecific communication. Fluorescence patterns were typically associated with the eyes or the head, varying substantially even between species of the same genus. Moreover red fluorescence was particularly strong in fins that are involved in intraspecific signalling. Finally, microspectrometry in one fluorescent goby, Eviota pellucida, showed a long-wave sensitivity that overlapped with its own red fluorescence, indicating that this species is capable of seeing its own fluorescence. Conclusion We show that red fluorescence is widespread among marine fishes. Many features indicate that it is used as a private communication mechanism in small, benthic, pair- or group-living fishes. Many of these species show quite cryptic colouration in other parts of the visible spectrum. High inter-specific variation in red fluorescence and its association with structures used in intra-specific signalling further corroborate this view. Our findings challenge the notion that red light is of no importance to marine fish, calling for a reassessment of its role in fish visual ecology in subsurface marine environments. PMID:18796150

In vivo three-photon microscopy of subcortical structures within an intact mouse brain

NASA Astrophysics Data System (ADS)

Horton, Nicholas G.; Wang, Ke; Kobat, Demirhan; Clark, Catharine G.; Wise, Frank W.; Schaffer, Chris B.; Xu, Chris

2013-03-01

Two-photon fluorescence microscopy enables scientists in various fields including neuroscience, embryology and oncology to visualize in vivo and ex vivo tissue morphology and physiology at a cellular level deep within scattering tissue. However, tissue scattering limits the maximum imaging depth of two-photon fluorescence microscopy to the cortical layer within mouse brain, and imaging subcortical structures currently requires the removal of overlying brain tissue or the insertion of optical probes. Here, we demonstrate non-invasive, high-resolution, in vivo imaging of subcortical structures within an intact mouse brain using three-photon fluorescence microscopy at a spectral excitation window of 1,700 nm. Vascular structures as well as red fluorescent protein-labelled neurons within the mouse hippocampus are imaged. The combination of the long excitation wavelength and the higher-order nonlinear excitation overcomes the limitations of two-photon fluorescence microscopy, enabling biological investigations to take place at a greater depth within tissue.

Advances in engineering of fluorescent proteins and photoactivatable proteins with red emission.

PubMed

Piatkevich, Kiryl D; Verkhusha, Vladislav V

2010-02-01

Monomeric fluorescent proteins of different colors are widely used to study behavior and targeting of proteins in living cells. Fluorescent proteins that irreversibly change their spectral properties in response to light irradiation of a specific wavelength, or photoactivate, have become increasingly popular to image intracellular dynamics and superresolution protein localization. Until recently, however, no optimized monomeric red fluorescent proteins and red photoactivatable proteins have been available. Furthermore, monomeric fluorescent proteins, which change emission from blue to red simply with time, so-called fluorescent timers, were developed to study protein age and turnover. Understanding of chemical mechanisms of the chromophore maturation or photoactivation into a red form will further advance engineering of fluorescent timers and photoactivatable proteins with enhanced and novel properties. 2009 Elsevier Ltd. All rights reserved.

Red light emitting nano-PVP fibers that hybrid with Ag@SiO2@Eu(tta)3phen-NPs by electrostatic spinning method

NASA Astrophysics Data System (ADS)

Zhang, Xiaolin; Tang, Jianguo; Li, Haidong; Wang, Yao; Wang, Xinzhi; Wang, Yanxin; Huang, Linjun; Belfiore, Laurence A.

2018-04-01

This work demonstrated red light emitting nano-PVP fibers that incorporated with novel three-layer nanostructure of Ag@SiO2@Eu(tta)3phen nanoparticles (Ag@SiO2@Eu(tta)3phen-NPs), and the hybrid nano-PVP fibers were fabricated via a remarkably simple electrostatic spinning method. For Ag@SiO2@Eu(tta)3phen-NPs, the thickness of SiO2 is optimized to obtain the maximum luminescent intensity, as results, the optimized thickness of SiO2 is 20 nm. And the corresponding luminescent intensity (612 nm) of the Ag@SiO2@Eu(tta)3phen-NPs is enhanced up to 10 times compared with the pure Eu(tta)3phen complex, which indicates that with 20 nm SiO2 thickness, the localized surface plasmon resonance (LSPR) effect of Ag@SiO2 exhibits highest performance for enhancing luminescence. Moreover, the luminescent PVP fibers emit bright red light under the fluorescence microscope, which definitely confirms that the microenvironment provided by PVP polymer is absolutely suitable for the fluorescent composites.

Guide to red fluorescent proteins and biosensors for flow cytometry.

PubMed

Piatkevich, Kiryl D; Verkhusha, Vladislav V

2011-01-01

Since the discovery of the first red fluorescent protein (RFP), named DsRed, 12 years ago, a wide pallet of red-shifted fluorescent proteins has been cloned and biotechnologically developed into monomeric fluorescent probes for optical microscopy. Several new types of monomeric RFPs that change the emission wavelength either with time, called fluorescent timers, or after a brief irradiation with violet light, known as photoactivatable proteins, have been also engineered. Moreover, RFPs with a large Stokes shift of fluorescence emission have been recently designed. Because of their distinctive excitation and fluorescence detection conditions developed specifically for microscopy, these fluorescent probes can be suboptimal for flow cytometry. Here, we have selected and summarized the advanced orange, red, and far-red fluorescent proteins with the properties specifically required for the flow cytometry applications. Their effective brightness was calculated for the laser sources available for the commercial flow cytometers and sorters. Compatibility of the fluorescent proteins of different colors in a multiparameter flow cytometry was determined. Novel FRET pairs, utilizing RFPs, RFP-based intracellular biosensors, and their application to a high-throughput screening, are also discussed. Copyright © 2011 Elsevier Inc. All rights reserved.

XRF and UV-Vis-NIR analyses of medieval wall paintings of al-Qarawiyyin Mosque (Morocco)

NASA Astrophysics Data System (ADS)

Fikri, I.; El Amraoui, M.; Haddad, M.; Ettahiri, A. S.; Bellot-Gurlet, L.; Falguères, C.; Lebon, M.; Nespoulet, R.; Ait Lyazidi, S.; Bejjit, L.

2018-05-01

Medieval wall painting fragments, taken at the medieval Mosque of al-Qarawiyyin in Fez, have been investigated by means of X-ray fluorescence and UV-Vis-NIR diffuse reflectance spectroscopies. The analyses permitted to determine the palette of pigments used by craftsmen of the time. Hematite or red ochre were used to obtain red brown colours, calcite for white, copper-based pigments for blue and blue-grey shades while a mixture of cinnabar, lead-based pigments and hematite was adopted to make red-orange colours. Furthermore, the analysis of mortars (external layer and plaster) on these wall painting samples revealed that they are composed mainly by calcite and sometimes by additional compounds such as quartz and gypsum.

Electroluminescence of fluorescent-phosphorescent organic light-emitting diodes with regular, inverted, and symmetrical structures

NASA Astrophysics Data System (ADS)

Yang, Su-Hua; Shih, Po-Jen; Wu, Wen-Jie

2014-11-01

The influence of the device structure on the electroluminescence (EL) properties of fluorescent-phosphorescent organic light emitting diodes (OLEDs) was demonstrated. Four devices with regular-, inverted-, compensated- and symmetrical-emission layers (EMLs) were prepared. In regular-EML device, DCJTB emission increased when the phosphorescent sensitized EML was thickened. In inverted-EML device, low electron energy barrier at the Bphen/BCzVB interface resulted in weakened blue emission. The compensated-EML device, prepared with a red color-compensated layer, showed a color-tunable broadband white emission. Conversely, device with a quantum-like symmetrical-EML showed a narrow color-temperature range. Stable EL efficiency was obtained from regular, compensated, and symmetrical-EML devices. In contrast, EL efficiency of inverted-EML device rolled off significantly, though it had the highest EL efficiency of 11.4 cd/A.

Spectroscopy detection of green and red fluorescent proteins in genetically modified plants using a fiber optics system

NASA Astrophysics Data System (ADS)

Liew, Oi Wah; Asundi, Anand K.; Chen, Jun-Wei; Chew, Yiwen; Yu, Shangjuan; Yeo, Gare H.

2001-05-01

In this paper, fiber optic spectroscopy is developed to detect and quantify recombinant green (EGFP) and red (DsRED) fluorescent proteins in vitro and in vivo. The bacterial expression vectors carrying the coding regions of EGFP and DsRED were introduced into Escherichia coli host cells and fluorescent proteins were produced following induction with IPTG. Soluble EGFP and DsRED proteins were isolated from lysed bacterial cells and serially diluted for quantitative analysis by fiber optic spectroscopy. Fluorescence at the appropriate emission wavelengths could be detected up to 64X dilution for EGFP and 40X dilution for DsRED. To determine the capability of spectroscopy detection in vivo, transgenic potato hairy roots expressing EGFP and DsRED were regenerated. This was achieved by cloning the EGFP and DsRED genes into the plant binary vector, pTMV35S, to create the recombinant vectors pGLOWGreen and pGLOWRed. These latter binary vectors were introduced into Agrobacterium rhizogenes strain A4T. Infection of potato cells with transformed agrobacteria was used to insert the fluorescent protein genes into the potato genome. Genetically modified potato cells were then regenerated into hairy roots. A panel of transformed hairy roots expressing varying levels of fluorescent proteins was selected by fluorescence microscopy. We are now assessing the capability of spectroscopic detection system for in vivo quantification of green and red fluorescence levels in transformed roots.

Development of practical red fluorescent probe for cytoplasmic calcium ions with greatly improved cell-membrane permeability.

PubMed

Hirabayashi, Kazuhisa; Hanaoka, Kenjiro; Egawa, Takahiro; Kobayashi, Chiaki; Takahashi, Shodai; Komatsu, Toru; Ueno, Tasuku; Terai, Takuya; Ikegaya, Yuji; Nagano, Tetsuo; Urano, Yasuteru

2016-10-01

Fluorescence imaging of calcium ions (Ca(2+)) has become an essential technique for investigation of signaling pathways involving Ca(2+) as a second messenger. But, Ca(2+) signaling is involved in many biological phenomena, and therefore simultaneous visualization of Ca(2+) and other biomolecules (multicolor imaging) would be particularly informative. For this purpose, we set out to develop a fluorescent probe for Ca(2+) that would operate in a different color region (red) from that of probes for other molecules, many of which show green fluorescence, as exemplified by green fluorescent protein (GFP). We previously developed a red fluorescent probe for monitoring cytoplasmic Ca(2+) concentration, based on our established red fluorophore, TokyoMagenta (TM), but there remained room for improvement, especially as regards efficiency of introduction into cells. We considered that this issue was probably mainly due to limited water solubility of the probe. So, we designed and synthesized a red-fluorescent probe with improved water solubility. We confirmed that this Ca(2+) red-fluorescent probe showed high cell-membrane permeability with bright fluorescence. It was successfully applied to fluorescence imaging of not only live cells, but also brain slices, and should be practically useful for multicolor imaging studies of biological mechanisms. Copyright © 2016 Elsevier Ltd. All rights reserved.

[Preparation, characterization and upconversion fluorescence of NaYF4 : Yb, Er /graphene oxide nanocomposites].

PubMed

Ji, Tian-Hao; Qie, Nan; Wang, Ji-Mei; Hua, Yong-Yong; Ji, Zhi-Jiang

2013-03-01

NaYF4 : Yb, Er/rGO and SiO2-coated NaYF4 : Yb, Er/rGO nanocomposites can be prepared through "one-pot" and directly mixing preparation routes. Various measurement results show that the NaYF4 : Yb, Er in the nanocomposites exhibits a cubic a-type structure and nanoparticle-like morphology with a diameter range of 30-70 nm; the rGO layers are well-dispersed in the nanocomposites, and whereas the rGO obtained from "one-pot" preparation renders relatively better dispersion. Raman spectra demonstrate that there exists a surface coupling action between the two kinds of nanomaterials, and with the increase in the relative rGO content, such action becomes stronger. UC fluorescence measurement results reveal that the rGO has significantly quenching effect and optical-limiting performance on the UC fluorescence, particularly on the red-emission of the NaYFa : Yb, Er or SiO2-coated NaYF4 : Yb, Er nanoparticles. The red-emission intensity gradually decreases with an increase in the rGO content, but the green-emission shows less change. It should be stressed that, in comparison with NaYF4 : Yb, Er/rGO, with a similar rGO content, the red-emission intensity of SiO2-coated NaYF4 : Yb, Er/rGO decreases much obviously due to a stronger light-absorption caused by part rGO aggregation.

Deep UV Native Fluorescence Imaging of Antarctic Cryptoendolithic Communities

NASA Technical Reports Server (NTRS)

Storrie-Lombardi, M. C.; Douglas, S.; Sun, H.; McDonald, G. D.; Bhartia, R.; Nealson, K. H.; Hug, W. F.

2001-01-01

An interdisciplinary team at the Jet Propulsion Laboratory Center for Life Detection has embarked on a project to provide in situ chemical and morphological characterization of Antarctic cryptoendolithic microbial communities. We present here in situ deep ultraviolet (UV) native fluorescence and environmental scanning electron microscopy images transiting 8.5 mm into a sandstone sample from the Antarctic Dry Valleys. The deep ultraviolet imaging system employs 224.3, 248.6, and 325 nm lasers to elicit differential fluorescence and resonance Raman responses from biomolecules and minerals. The 224.3 and 248.6 nm lasers elicit a fluorescence response from the aromatic amino and nucleic acids. Excitation at 325 nm may elicit activity from a variety of biomolecules, but is more likely to elicit mineral fluorescence. The resultant fluorescence images provide in situ chemical and morphological maps of microorganisms and the associated organic matrix. Visible broadband reflectance images provide orientation against the mineral background. Environmental scanning electron micrographs provided detailed morphological information. The technique has made possible the construction of detailed fluorescent maps extending from the surface of an Antarctic sandstone sample to a depth of 8.5 mm. The images detect no evidence of microbial life in the superficial 0.2 mm crustal layer. The black lichen component between 0.3 and 0.5 mm deep absorbs all wavelengths of both laser and broadband illumination. Filamentous deep ultraviolet native fluorescent activity dominates in the white layer between 0.6 mm and 5.0 mm from the surface. These filamentous forms are fungi that continue into the red (iron-rich) region of the sample extending from 5.0 to 8.5 mm. Using differential image subtraction techniques it is possible to identify fungal nuclei. The ultraviolet response is markedly attenuated in this region, apparently from the absorption of ultraviolet light by iron-rich particles coating the filaments. Below 8.5 mm the filamentous morphology of the upper layers gives way to punctate 1-2 micron particles evidencing fluorescent activity following excitation at both deep ultraviolet wavelengths.

Red fluorescence of the triplefin Tripterygion delaisi is increasingly visible against background light with increasing depth.

PubMed

Bitton, Pierre-Paul; Harant, Ulrike K; Fritsch, Roland; Champ, Connor M; Temple, Shelby E; Michiels, Nico K

2017-03-01

The light environment in water bodies changes with depth due to the absorption of short and long wavelengths. Below 10 m depth, red wavelengths are almost completely absent rendering any red-reflecting animal dark and achromatic. However, fluorescence may produce red coloration even when red light is not available for reflection. A large number of marine taxa including over 270 fish species are known to produce red fluorescence, yet it is unclear under which natural light environment fluorescence contributes perceptively to their colours. To address this question we: (i) characterized the visual system of Tripterygion delaisi, which possesses fluorescent irides, (ii) separated the colour of the irides into its reflectance and fluorescence components and (iii) combined these data with field measurements of the ambient light environment to calculate depth-dependent perceptual chromatic and achromatic contrasts using visual modelling. We found that triplefins have cones with at least three different spectral sensitivities, including differences between the two members of the double cones, giving them the potential for trichromatic colour vision. We also show that fluorescence contributes increasingly to the radiance of the irides with increasing depth. Our results support the potential functionality of red fluorescence, including communicative roles such as species and sex identity, and non-communicative roles such as camouflage.

Structure-guided wavelength tuning in far-red fluorescent proteins

PubMed Central

Ng, Ho-Leung; Lin, Michael Z.

2017-01-01

In recent years, protein engineers have succeeded in tuning the excitation spectra of natural fluorescent proteins from green wavelengths into orange and red wavelengths, resulting in the creation of a series of fluorescent proteins with emission in the far-red portions of the optical spectrum. These results have arisen from the synergistic combination of structural knowledge of fluorescent proteins, chemical intuition, and high-throughput screening methods. Here we review structural features found in autocatalytic far-red fluorescent proteins, and discuss how they add to our understanding of the biophysical mechanisms of wavelength tuning in biological chromophores. PMID:27468111

A Novel Mechanism for the Pathogenesis of Nonmelanoma Skin Cancer Resulting from Early Exposure to Ultraviolet Light

DTIC Science & Technology

2014-09-01

hybrid mice show a large population of cells that fluoresce with Tomato Red and few cells that fluoresce with GFP only or GFP/ Tomato Red double positive...percent of total cells Double Negative GFP Tomato Red Double Positive 15 Figure 3. Fluorescent activated cell sorting (FACS) shows slight...Negative Tomato Red Double Positive 17 Figure 5. Fluorescent activated cell sorting (FACS) shows no K14-GFP expressing cells and slight expression of

Expression of fluorescently tagged connexins: a novel approach to rescue function of oligomeric DsRed-tagged proteins.

PubMed

Lauf, U; Lopez, P; Falk, M M

2001-06-01

A novel, brilliantly red fluorescent protein, DsRed has become available recently opening up a wide variety of experimental opportunities for double labeling and fluorescence resonance electron transfer experiments in combination with green fluorescent protein (GFP). Unlike in the case of GFP, proteins tagged with DsRed were often found to aggregate within the cell. Here we report a simple method that allows rescuing the function of an oligomeric protein tagged with DsRed. We demonstrate the feasibility of this approach on the subunit proteins of an oligomeric membrane channel, gap junction connexins. Additionally, DsRed fluorescence was easily detected 12-16 h post transfection, much earlier than previously reported, and could readily be differentiated from co-expressed GFP. Thus, this approach can eliminate the major drawbacks of this highly attractive autofluorescent protein.

Stably Fluorescent Cell Line of Human Ovarian Epithelial Cancer Cells SK-OV-3ip-red.

PubMed

Konovalova, E V; Shulga, A A; Chumakov, S P; Khodarovich, Yu M; Woo, Eui-Jeon; Deev, S M

2017-11-01

Stable red fluorescing line of human ovarian epithelial cancer cells SK-OV-3ip-red was generated expressing gene coding for protein TurboFP635 (Katushka) fluorescing in the far-red spectrum region with excitation and emission peaks at 588 and 635 nm, respectively. Fluorescence of SK-OV-3ip-red line remained high during long-term cell culturing and after cryogenic freezing. The obtained cell line SK-OV-3ip-red can serve a basis for a model of a scattered tumor with numerous/extended metastases and used both for testing anticancer drugs inhibiting metastasis growth and for non-invasive monitoring of the growth dynamics with high precision.

Cracks in the beta-can: fluorescent proteins from Anemonia sulcata (Anthozoa, Actinaria).

PubMed

Wiedenmann, J; Elke, C; Spindler, K D; Funke, W

2000-12-19

We characterize two green fluorescent proteins (GFPs), an orange fluorescent protein, and a nonfluorescent red protein isolated from the sea anemone Anemonia sulcata. The orange fluorescent protein and the red protein seem to represent two different states of the same protein. Furthermore, we describe the cloning of a GFP and a nonfluorescent red protein. Both proteins are homologous to the GFP from Aequorea victoria. The red protein is significantly smaller than other GFP homologues, and the formation of a closed GFP-like beta-can is not possible. Nevertheless, the primary structure of the red protein carries all features necessary for orange fluorescence. We discuss a type of beta-can that could be formed in a multimerization process.

Cracks in the β-can: Fluorescent proteins from Anemonia sulcata (Anthozoa, Actinaria)

PubMed Central

Wiedenmann, Jörg; Elke, Carsten; Spindler, Klaus-Dieter; Funke, Werner

2000-01-01

We characterize two green fluorescent proteins (GFPs), an orange fluorescent protein, and a nonfluorescent red protein isolated from the sea anemone Anemonia sulcata. The orange fluorescent protein and the red protein seem to represent two different states of the same protein. Furthermore, we describe the cloning of a GFP and a nonfluorescent red protein. Both proteins are homologous to the GFP from Aequorea victoria. The red protein is significantly smaller than other GFP homologues, and the formation of a closed GFP-like β-can is not possible. Nevertheless, the primary structure of the red protein carries all features necessary for orange fluorescence. We discuss a type of β-can that could be formed in a multimerization process. PMID:11121018

Effect of horizontal molecular orientation on triplet-exciton diffusion in amorphous organic films

NASA Astrophysics Data System (ADS)

Sawabe, T.; Takasu, I.; Yonehara, T.; Ono, T.; Yoshida, J.; Enomoto, S.; Amemiya, I.; Adachi, C.

2012-09-01

Triplet harvesting is a candidate technology for highly efficient and long-life white OLEDs, where green or red phosphorescent emitters are activated by the triplet-excitons diffused from blue fluorescent emitters. We examined two oxadiazole-based electron transport materials with different horizontal molecular orientation as a triplet-exciton diffusion layer (TDL) in triplet-harvesting OLEDs. The device characteristics and the transient electroluminescent analyses of the red phosphorescent emitter showed that the triplet-exciton diffusion was more effective in the highly oriented TDL. The results are ascribed to the strong orbital overlap between the oriented molecules, which provides rapid electron exchange (Dexter energy transfer) in the TDL.

Confinement of carbon dots localizing to the ultrathin layered double hydroxides toward simultaneous triple-mode bioimaging and photothermal therapy.

PubMed

Weng, Yangziwan; Guan, Shanyue; Lu, Heng; Meng, Xiangmin; Kaassis, Abdessamad Y; Ren, Xiaoxue; Qu, Xiaozhong; Sun, Chenghua; Xie, Zheng; Zhou, Shuyun

2018-07-01

It is a great challenge to develop multifunctional nanocarriers for cancer diagnosis and therapy. Herein, versatile CDs/ICG-uLDHs nanovehicles for triple-modal fluorescence/photoacoustic/two-photon bioimaging and effective photothermal therapy were prepared via a facile self-assembly of red emission carbon dots (CDs), indocyanine green (ICG) with the ultrathin layered double hydroxides (uLDHs). Due to the J-aggregates of ICG constructed in the self-assembly process, CDs/ICG-uLDHs was able to stabilize the photothermal agent ICG and enhanced its photothermal efficiency. Furthermore, the unique confinement effect of uLDHs has extended the fluorescence lifetime of CDs in favor of bioimaging. Considering the excellent in vitro and in vivo phototherapeutics and multimodal imaging effects, this work provides a promising platform for the construction of multifunctional theranostic nanocarrier system for the cancer treatment. Copyright © 2018 Elsevier B.V. All rights reserved.

Red fluorescence increases with depth in reef fishes, supporting a visual function, not UV protection

PubMed Central

Meadows, Melissa G.; Anthes, Nils; Dangelmayer, Sandra; Alwany, Magdy A.; Gerlach, Tobias; Schulte, Gregor; Sprenger, Dennis; Theobald, Jennifer; Michiels, Nico K.

2014-01-01

Why do some marine fishes exhibit striking patterns of natural red fluorescence? In this study, we contrast two non-exclusive hypotheses: (i) that UV absorption by fluorescent pigments offers significant photoprotection in shallow water, where UV irradiance is strongest; and (ii) that red fluorescence enhances visual contrast at depths below −10 m, where most light in the ‘red’ 600–700 nm range has been absorbed. Whereas the photoprotection hypothesis predicts fluorescence to be stronger near the surface and weaker in deeper water, the visual contrast hypothesis predicts the opposite. We used fluorometry to measure red fluorescence brightness in vivo in individuals belonging to eight common small reef fish species with conspicuously red fluorescent eyes. Fluorescence was significantly brighter in specimens from the −20 m sites than in those from −5 m sites in six out of eight species. No difference was found in the remaining two. Our results support the visual contrast hypothesis. We discuss the possible roles fluorescence may play in fish visual ecology and highlight the possibility that fluorescent light emission from the eyes in particular may be used to detect cryptic prey. PMID:25030989

A direct and simultaneous detection of zinc protoporphyrin IX, free protoporphyrin IX, and fluorescent heme degradation product in red blood cell hemolysates.

PubMed

Chen, Qiuying; Hirsch, Rhoda Elison

2006-03-01

Fluorescence emission of free protoporphyrin IX (PPIX, em. approximately 626 nm), zinc protoporphyrin IX (ZPP, em. approximately 594 nm) and fluorescent heme degradation product (FHDP, em. approximately 466 nm) are identified and simultaneously detected in mouse and human red cell hemolysates, when excited at 365 nm. A novel method is established for comparing relative FHDP, PPIX and ZPP levels in hemolysates without performing red cell porphyrin extractions. The ZPP fluorescence directly measured in hemolysates (F(365/594)) correlates with the ZPP fluorescence obtained from acetone/water extraction (R(2) = 0.9515, P < 0.0001). The relative total porphyrin (ZPP and PPIX) fluorescence obtained from direct hemolysate fluorescence measurements also correlates with red blood cell total porphyrins determined by ethyl acetate extraction (Piomelli extraction, R(2) = 0.88, P < 0.0001). These fluorescent species serves as biomarkers for alterations in Hb synthesis and Hb stability.

Light-induced flickering of DsRed provides evidence for distinct and interconvertible fluorescent states.

PubMed Central

Malvezzi-Campeggi, F; Jahnz, M; Heinze, K G; Dittrich, P; Schwille, P

2001-01-01

Green fluorescent protein (GFP) from jellyfish Aequorea victoria, the powerful genetically encoded tag presently available in a variety of mutants featuring blue to yellow emission, has found a red-emitting counterpart. The recently cloned red fluorescent protein DsRed, isolated from Discosoma corals (), with its emission maximum at 583 nm, appears to be the long awaited tool for multi-color applications in fluorescence-based biological research. Studying the emission dynamics of DsRed by fluorescence correlation spectroscopy (FCS), it can be verified that this protein exhibits strong light-dependent flickering similar to what is observed in several yellow-shifted mutants of GFP. FCS data recorded at different intensities and excitation wavelengths suggest that DsRed appears under equilibrated conditions in at minimum three interconvertible states, apparently fluorescent with different excitation and emission properties. Light absorption induces transitions and/or cycling between these states on time scales of several tens to several hundreds of microseconds, dependent on excitation intensity. With increasing intensity, the emission maximum of the static fluorescence continuously shifts to the red, implying that at least one state emitting at longer wavelength is preferably populated at higher light levels. In close resemblance to GFP, this light-induced dynamic behavior implies that the chromophore is subject to conformational rearrangements upon population of the excited state. PMID:11509387

A pH-sensitive red fluorescent protein compatible with hydrophobic resin embedding

NASA Astrophysics Data System (ADS)

Guo, Wenyan; Gang, Yadong; Liu, Xiuli; Zhou, Hongfu; Zeng, Shaoqun

2017-02-01

pH sensitive fluorescent proteins enabling chemical reactivation in resin are useful tools for fluorescence microimaging. EYFP or EGFP improved from GFP in jellyfish are good for such applications. For simultaneous two-color imaging, a suitable red fluorescent protein is of urgent need. Here a pH sensitive red fluorescent protein, pHuji, is selected and verified to be compatible with hydrophobic resin embedding and thus may be promising for dual-colour chemical reactivation imaging in conjunction with EGFP or EYFP.

Evaluation of chemical fluorescent dyes as a protein conjugation partner for live cell imaging.

PubMed

Hayashi-Takanaka, Yoko; Stasevich, Timothy J; Kurumizaka, Hitoshi; Nozaki, Naohito; Kimura, Hiroshi

2014-01-01

To optimize live cell fluorescence imaging, the choice of fluorescent substrate is a critical factor. Although genetically encoded fluorescent proteins have been used widely, chemical fluorescent dyes are still useful when conjugated to proteins or ligands. However, little information is available for the suitability of different fluorescent dyes for live imaging. We here systematically analyzed the property of a number of commercial fluorescent dyes when conjugated with antigen-binding (Fab) fragments directed against specific histone modifications, in particular, phosphorylated H3S28 (H3S28ph) and acetylated H3K9 (H3K9ac). These Fab fragments were conjugated with a fluorescent dye and loaded into living HeLa cells. H3S28ph-specific Fab fragments were expected to be enriched in condensed chromosomes, as H3S28 is phosphorylated during mitosis. However, the degree of Fab fragment enrichment on mitotic chromosomes varied depending on the conjugated dye. In general, green fluorescent dyes showed higher enrichment, compared to red and far-red fluorescent dyes, even when dye:protein conjugation ratios were similar. These differences are partly explained by an altered affinity of Fab fragment after dye-conjugation; some dyes have less effect on the affinity, while others can affect it more. Moreover, red and far-red fluorescent dyes tended to form aggregates in the cytoplasm. Similar results were observed when H3K9ac-specific Fab fragments were used, suggesting that the properties of each dye affect different Fab fragments similarly. According to our analysis, conjugation with green fluorescent dyes, like Alexa Fluor 488 and Dylight 488, has the least effect on Fab affinity and is the best for live cell imaging, although these dyes are less photostable than red fluorescent dyes. When multicolor imaging is required, we recommend the following dye combinations for optimal results: Alexa Fluor 488 (green), Cy3 (red), and Cy5 or CF640 (far-red).

[Establishment and identification of mouse lymphoma cell line EL4 expressing red fluorescent protein].

PubMed

Li, Yan-Jie; Cao, Jiang; Chen, Chong; Wang, Dong-Yang; Zeng, Ling-Yu; Pan, Xiu-Ying; Xu, Kai-Lin

2010-02-01

This study was purposed to construct a lentiviral vector encoding red fluorescent protein (DsRed) and transfect DsRed into EL4 cells for establishing mouse leukemia/lymphoma model expressing DsRed. The bicistronic SIN lentiviral transfer plasmid containing the genes encoding neo and internal ribosomal entry site-red fluorescent protein (IRES-DsRed) was constructed. Human embryonic kidney 293FT cells were co-transfected with the three plasmids by liposome method. The viral particles were collected and used to transfect EL4 cells, then the cells were selected by G418. The results showed that the plasmid pXZ208-neo-IRES-DsRed was constructed successfully, and the viral titer reached to 10(6) U/ml. EL4 cells were transfected by the viral solution efficiently. The transfected EL4 cells expressing DsRed survived in the final concentration 600 microg/ml of G418. The expression of DsRed in the transfected EL4 cells was demonstrated by fluorescence microscopy and flow cytometry. In conclusion, the EL4/DsRed cell line was established successfully.

Investigating the Modification of Spontaneous Emission using Layer-by-Layer Self-Assembly

NASA Astrophysics Data System (ADS)

Ashry, Islam Ahmed Ibrahim Youssef

The process of spontaneous emission can be dramatically modified by optical micro- and nanostructures. We studied the modification of fluorescence dynamics using a polymer spacer layer fabricated through layer-by-layer (LbL) self-assembly. The advantages of this method are numerous: The self-assembled spacers can possess exceptional smooth surface morphology; The thickness of the spacer can be controlled with nanometer accuracy; And depending on fabrication conditions, the spacer layer is stimuli responsive and its thickness can be dynamically tuned. This thesis contains three interlinked components. First, we vary LbL spacer layer thickness and explore the change in fluorescence lifetime induced by the modified photonic density of states (PDOS), i.e., Purcell effects. Our experimental results agree well with theoretical predictions based on a classical dipole model, which also yields consistent values for the fluorophores' intrinsic fluorescence lifetime and quantum yield near a dielectric as well as a plasmonic interface. Based on this observation, we further demonstrate that self-assembled fluorophores can be used to probe the modified PDOS near optical micro- and nano-structures. These results naturally lead to the second component of our research. In particularly, based on the PDOS-induced changes in fluorescent lifetime, we develop a non-contact method that can measure morphological changes with nanoscale resolution. Our method relies on quantitatively linking fluorophore position with PDOS, and is validated through direct comparison with ellipsometry and atomic force microscopy (AFM) measurements. To demonstrate the potential application of this method, we investigated the swelling/deswelling of LbL films induced by pH changes. Our results indicate significant difference between a LbL film composed of a single polymer monolayer and a LbL film with 3 monolayers. Such stimuli-responsive polymers can be used to construct active and tunable plasmonic nano-devices. As a proof-of-principle demonstration, we experimentally confirm that it is possible to utilize the swelling/deswelling behavior of stimuli-responsive films to dynamically control the separation between Au nanoparticles and Texas Red (TR) dyes. This result is based on the strong correlation of TR fluorescence lifetime and nanoparticles-TR separation. Finally, we investigate the impact of different lithography processes on the fluorescence properties of self-assembled fluorophores. We consider three methods: direct fluorophore patterning through ultraviolet (UV) ablation, focused ion beam (FIB) milling of self-assembled fluorophores, and self-assembly of fluorescent materials over plasmonic nano-patterns.

Observation of Zn-photoprotoporphyrin red Autofluorescence in human bronchial cancer using color-fluorescence endoscopy.

PubMed

Ohsaki, Yoshinobu; Sasaki, Takaaki; Endo, Satoshi; Kitada, Masahiro; Okumura, Shunsuke; Hirai, Noriko; Kazebayashi, Yoshihiro; Toyoshima, Eri; Yamamoto, Yasushi; Takeyama, Kaneyoshi; Nakajima, Susumu; Sakata, Isao

2017-04-26

We observed red autofluorescence emanating from bronchial cancer lesions using a sensitive color-fluorescence endoscopy system. We investigated to clarify the origin of the red autofluorescence. The wavelengths of the red autofluorescence emanating from lesions were measured in eight patients using a spectrum analyzer and compared based on pathologic findings. Red autofluorescence at 617.3, 617.4, 619.0, and 617.1 nm was emitted by normal bronchus, inflamed tissue, tissue exhibiting mild dysplasia, and malignant lesions, respectively. Protoporphyrin, uroporphyrin, and coproporphyrin, the major porphyrin derivatives in human blood, were purchased to determine which porphyrin derivative is the source of red fluorescence when acquired de novo. We synthesized photoporphyrin, Zn-protoporphyrin and Zn-photoprotoporphyrin from protoporphyrin. Coproporphyrin and uroporphyrin emitted only weak fluorescence. Fluorescence was emitted by our synthesized Zn-photoprotoporphyrin at 625.5 nm and by photoprotoporphyrin at 664.0 nm. From these results, we conclude that Zn-photoprotoporphyrin was the source of the red autofluorescence observed in bronchial lesions. Zn-protoporphyrin is converted to Zn-photoprotoporphyrin by radiation with excitation light. Our results suggest that red autofluorescence emanating from Zn-photoprotoporphyrin in human tissues could interfere with photodynamic diagnosis using porphyrin derivatives such as Photofrin® and Lazerphyrin® with a sensitive endoscopy system, because color cameras cannot differentiate Zn-photoprotoporphyrin red fluorescence from that of other porphyrin derivatives.

Cytokeratin expression of engrafted three-dimensional culture tissues using epithelial cells derived from porcine periodontal ligaments.

PubMed

Yamada, Rie; Kitajima, Kayoko; Arai, Kyoko; Igarashi, Masaru

2014-09-01

This study investigated the differentiation and proliferation of epithelial cells derived from periodontal ligaments after three-dimensional culture using collagen gel with fibroblasts in vitro and in vivo. Epithelial cells and fibroblasts were derived from porcine periodontal ligaments. Epithelial cells were labeled using a fluorescent red membrane marker (PKH-26GL) and were seeded onto collagen gel with fibroblasts, followed by incubation in an air-liquid interface for 7 days. Three-dimensional cultures were grafted onto the backs of nude mice and removed at 1, 7, and 14 days after surgery (in vivo model). Unfixed sections (5 μm) were used to detect the presence of red fluorescent cells. Paraffin sections were analyzed histologically and immunohistochemically. Specimens were compared with three-dimensional culture tissues at 8, 14 and 21 days (in vitro model). Grafted three-dimensional cultures formed a stratified epithelial structure similar to skin in vivo. Epithelial cells were sequenced in basal-layer-like structures at 14 days in vivo. Immunohistochemical findings showed that the expression of cytokeratin was detected in the epithelial layer in in vitro and in vivo models. Ck8 + 18 + 19 was expressed in the upper epithelial layer in the in vitro model at 14 and 21 days, but not in vivo. Involucrin was expressed in the certified layers in vitro at 14 days, but not in vivo. Laminin was detected at the dermo-epidermal junction in vivo at 7 and 14 days, but not in vitro. These results suggest that differentiation of three-dimensional culture tissues differs in vivo and in vitro. © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Analytical investigation of Mudéjar polychrome on the carpentry in the Casa de Pilatos palace in Seville using non-destructive XRF and complementary techniques

NASA Astrophysics Data System (ADS)

Garrote, M. A.; Robador, M. D.; Perez-Rodriguez, J. L.

2017-02-01

The pigments, execution technique and repainting used on the polychrome wood ceilings and doors in the Casa de Pilatos (Seville, Spain) were studied using portable X-ray fluorescence equipment. Cross-sections of small samples were also analysed by optical microscopy, SEM with EDX analysis, micro-Raman and micro-infrared spectroscopy and X-ray diffraction. These carpentry works are magnificent examples of the Mudéjar art made in Spain in the early 16th century. Portable X-ray fluorescence gave good information on the different components of the polychrome. The SEM-EDX study of the surfaces of small samples gave information on their components and also characterized the compounds that had been deposited or formed by environmental contamination or by the alteration of some pigments. The SEM-EDX study of cross-sections facilitated the characterization of all layers and pigments from the support to the most external layer. The following pigments were characterized: red (cinnabar/vermillion, lead oxide, iron oxides and orpiment/realgar), black (carbon black), white (white lead and titanium barium white), yellow-orange-red-brown (orpiment/realgar and iron oxides), green (chromium oxide), blue (indigo blue and ultramarine blue), and gilding (gold leaf on bole). False gold, bronze and brass were also found. The pigments were applied with the oil painting technique over a support layer that had been primed with animal glue. This support layer was gypsum in some cases and white lead in others. This study is essential to the polychrome conservation of the studied artwork, and it will help clarify uncertainties in the history and painting of Mudéjar art.

Analytical investigation of Mudéjar polychrome on the carpentry in the Casa de Pilatos palace in Seville using non-destructive XRF and complementary techniques.

PubMed

Garrote, M A; Robador, M D; Perez-Rodriguez, J L

2017-02-15

The pigments, execution technique and repainting used on the polychrome wood ceilings and doors in the Casa de Pilatos (Seville, Spain) were studied using portable X-ray fluorescence equipment. Cross-sections of small samples were also analysed by optical microscopy, SEM with EDX analysis, micro-Raman and micro-infrared spectroscopy and X-ray diffraction. These carpentry works are magnificent examples of the Mudéjar art made in Spain in the early 16th century. Portable X-ray fluorescence gave good information on the different components of the polychrome. The SEM-EDX study of the surfaces of small samples gave information on their components and also characterized the compounds that had been deposited or formed by environmental contamination or by the alteration of some pigments. The SEM-EDX study of cross-sections facilitated the characterization of all layers and pigments from the support to the most external layer. The following pigments were characterized: red (cinnabar/vermillion, lead oxide, iron oxides and orpiment/realgar), black (carbon black), white (white lead and titanium barium white), yellow-orange-red-brown (orpiment/realgar and iron oxides), green (chromium oxide), blue (indigo blue and ultramarine blue), and gilding (gold leaf on bole). False gold, bronze and brass were also found. The pigments were applied with the oil painting technique over a support layer that had been primed with animal glue. This support layer was gypsum in some cases and white lead in others. This study is essential to the polychrome conservation of the studied artwork, and it will help clarify uncertainties in the history and painting of Mudéjar art. Copyright © 2016 Elsevier B.V. All rights reserved.

Development of a Convenient In Vivo Hepatotoxin Assay Using a Transgenic Zebrafish Line with Liver-Specific DsRed Expression

PubMed Central

Zhang, Xiaoyan; Li, Caixia; Gong, Zhiyuan

2014-01-01

Previously we have developed a transgenic zebrafish line (LiPan) with liver-specific red fluorescent protein (DsRed) expression under the fabp10a promoter. Since red fluorescence in the liver greatly facilitates the observation of liver in live LiPan fry, we envision that the LiPan zebrafish may provide a useful tool in analyses of hepatotoxicity based on changes of liver red fluorescence intensity and size. In this study, we first tested four well-established hepatotoxins (acetaminophen, aspirin, isoniazid and phenylbutazone) in LiPan fry and demonstrated that these hepatotoxins could significantly reduce both liver red fluorescence and liver size in a dosage-dependent manner, thus the two measurable parameters could be used as indicators of hepatotoxicity. We then tested the LiPan fry with nine other chemicals including environmental toxicants and human drugs. Three (mefenamic acid, lindane, and arsenate) behave like hepatotoxins in reduction of liver red fluorescence, while three others (17β-estradiol, TCDD [2,3,7,8-tetrachlorodibenzo-p-dioxin] and NDMA [N-nitrosodimethylamine]) caused increase of liver red fluorescence and the liver size. Ethanol and two other chemicals, amoxicillin (antibiotics) and chlorphenamine (pain killer) did not resulted in significant changes of liver red fluorescence and liver size. By quantitative RT-PCR analysis, we found that the changes of red fluorescence intensity caused by different chemicals correlated to the changes of endogenous fabp10a RNA expression, indicating that the measured hepatotoxicity was related to fatty acid transportation and metabolism. Finally we tested a mixture of four hepatotoxins and observed a significant reduction of red fluorescence in the liver at concentrations below the lowest effective concentrations of individual hepatotoxins, suggesting that the transgenic zebrafish assay is capable of reporting compound hepatotoxicity effect from chemical mixtures. Thus, the LiPan transgenic fry provide a rapid and convenient in vivo hepatotoxicity assay that should be applicable to high-throughput hepatotoxicity test in drug screening as well as in biomonitoring environmental toxicants. PMID:24626481

[Applications of three-dimensional fluorescence spectrum of dissolved organic matter to identification of red tide algae].

PubMed

Lü, Gui-Cai; Zhao, Wei-Hong; Wang, Jiang-Tao

2011-01-01

The identification techniques for 10 species of red tide algae often found in the coastal areas of China were developed by combining the three-dimensional fluorescence spectra of fluorescence dissolved organic matter (FDOM) from the cultured red tide algae with principal component analysis. Based on the results of principal component analysis, the first principal component loading spectrum of three-dimensional fluorescence spectrum was chosen as the identification characteristic spectrum for red tide algae, and the phytoplankton fluorescence characteristic spectrum band was established. Then the 10 algae species were tested using Bayesian discriminant analysis with a correct identification rate of more than 92% for Pyrrophyta on the level of species, and that of more than 75% for Bacillariophyta on the level of genus in which the correct identification rates were more than 90% for the phaeodactylum and chaetoceros. The results showed that the identification techniques for 10 species of red tide algae based on the three-dimensional fluorescence spectra of FDOM from the cultured red tide algae and principal component analysis could work well.

Red fluorescence imaging for dental plaque detection and quantification: pilot study

NASA Astrophysics Data System (ADS)

Liu, Zhao; Gomez, Juliana; Khan, Soniya; Peru, Debbie; Ellwood, Roger

2017-09-01

The red fluorescence of dental plaque originating from porphyrins in oral bacteria may allow visualization, detection, and scoring of plaque without disclosing agents. Two studies were conducted. The first included 24 healthy participants who abstained from oral hygiene for 24 h. Dental plaque was collected from tooth surfaces, and a 10% solution was prepared. These were scanned by a molecular spectrometer to identify the optimum excitation and emission wavelengths of plaque for developing a red fluorescence imaging system. Fourteen healthy subjects completed the second study. After a washout period (1 week), participants had a prophylaxis at baseline and abstained from oral hygiene during the study. They were monitored using the fluorescence imaging system at baseline, 24 h, and 48 h. A dentist clinically assessed plaque after disclosing and on red fluorescence images. Three descriptors were extracted from images and a RUSBoost classifier derived computer fluorescence scores through cross-validation. Red fluorescence plaque levels increased during the 48-h accumulation. Plaque progression was identified by dentist assessment and computer analysis, presenting significant differences between visits at tooth and subject levels (p<0.05). Moderate correlations showed between clinical plaque and red fluorescence plaque (r=0.62 dentist, r=0.55 computer). The best agreement was observed when disclosing plaque threshold at level 2, for both dentist evaluation (sensitivity 71.1%, specificity 67.7%, accuracy 70.2%) and computer classification (sensitivity 68.4%, specificity 62.9%, accuracy 67.1%). Given the correlation with clinical diagnosis, red fluorescence imaging shows its potential for providing an objective and promising method for proper oral hygiene assessment.

Detection of silver nanoparticles in cells by flow cytometry using light scatter and far-red fluorescence.

PubMed

Zucker, R M; Daniel, K M; Massaro, E J; Karafas, S J; Degn, L L; Boyes, W K

2013-10-01

The cellular uptake of different sized silver nanoparticles (AgNP) (10, 50, and 75 nm) coated with polyvinylpyrrolidone (PVP) or citrate on a human derived retinal pigment epithelial cell line (ARPE-19) was detected by flow cytometry following 24-h incubation of the cells with AgNP. A dose dependent increase of side scatter and far red fluorescence was observed with both PVP and citrate-coated 50 nm or 75 nm silver particles. Using five different flow cytometers, a far red fluorescence signal in the 700-800 nm range increased as much as 100 times background as a ratio comparing the intensity measurements of treated sample and controls. The citrate-coated silver nanoparticles (AgNP) revealed slightly more side scatter and far red fluorescence than did the PVP coated silver nanoparticles. This increased far red fluorescence signal was observed with 50 and 75 nm particles, but not with 10 nm particles. Morphological evaluation by dark field microscopy showed silver particles (50 and 75 nm) clumped and concentrated around the nucleus. One possible hypothesis to explain the emission of far red fluorescence from cells incubated with silver nanoparticles is that the silver nanoparticles inside cells agglomerate into small nano clusters that form surface plasmon resonance which interacts with laser light to emit a strong far red fluorescence signal. The results demonstrate that two different parameters (side scatter and far red fluorescence) on standard flow cytometers can be used to detect and observe metallic nanoparticles inside cells. The strength of the far red fluorescence suggests that it may be particularly useful for applications that require high sensitivity. © Published 2013 Wiley-Periodicals, Inc. Published 2013 Wiley‐Periodicals, Inc. This article is a US government work and, as such, is in the public domain in the United States of America.

Novel techniques with multiphoton microscopy: Deep-brain imaging with microprisms, neurometabolism of epilepsy, and counterfeit paper money detection

NASA Astrophysics Data System (ADS)

Chia, Thomas H.

Multiphoton microscopy is a laser-scanning fluorescence imaging method with extraordinary potential. We describe three innovative multiphoton microscopy techniques across various disciplines. Traditional in vivo fluorescence microscopy of the mammalian brain has a limited penetration depth (<400 microm). We present a method of imaging 1 mm deep into mouse neocortex by using a glass microprism to relay the excitation and emission light. This technique enables simultaneous imaging of multiple cortical layers, including layer V, at an angle typical of slice preparations. At high-magnification imaging using an objective with 1-mm of coverglass correction, resolution was sufficient to resolve dendritic spines on layer V GFP neurons. Functional imaging of blood flow at various neocortical depths is also presented, allowing for quantification of red blood cell flux and velocity. Multiphoton fluorescence lifetime imaging (FLIM) of NADH reveals information on neurometabolism. NADH, an intrinsic fluorescent molecule and ubiquitous metabolic coenzyme, has a lifetime dependent on enzymatic binding. A novel NADH FLIM algorithm is presented that produces images showing spatially distinct NADH fluorescence lifetimes in mammalian brain slices. This program provides advantages over traditional FLIM processing of multi-component lifetime data. We applied this technique to a GFP-GFAP pilocarpine mouse model of temporal lobe epilepsy. Results indicated significant changes in the neurometabolism of astrocytes and neuropil in the cell and dendritic layers of the hippocampus when compared to control tissue. Data obtained with NADH FLIM were subsequently interpreted based on the abnormal activity reported in epileptic tissue. Genuine U.S. Federal Reserve Notes have a consistent, two-component intrinsic fluorescence lifetime. This allows for detection of counterfeit paper money because of its significant differences in fluorescence lifetime when compared to genuine paper money. We used scanning multiphoton laser excitation to sample a ˜4 mm2 region from 54 genuine Reserve Notes. Three types of counterfeit samples were tested. Four out of the nine counterfeit samples fit to a one-component decay. Five out of nine counterfeit samples fit to a two-component model, but are identified as counterfeit due to significant deviations in the longer lifetime component compared to genuine bills.

Environmental effects on underwater optical transmission

NASA Astrophysics Data System (ADS)

Chu, Peter C.; Breshears, Brian F.; Cullen, Alexander J.; Hammerer, Ross F.; Martinez, Ramon P.; Phung, Thai Q.; Margolina, Tetyana; Fan, Chenwu

2017-05-01

Optical communication/detection systems have potential to get around some limitations of current acoustic communications and detection systems especially increased fleet and port security in noisy littoral waters. Identification of environmental effects on underwater optical transmission is the key to the success of using optics for underwater communication and detection. This paper is to answer the question "What are the transfer and correlation functions that relate measurements of hydrographic to optical parameters?" Hydrographic and optical data have been collected from the Naval Oceanographic Office survey ships with the High Intake Defined Excitation (HIDEX) photometer and sea gliders with optical back scattering sensor in various Navy interested areas such as the Arabian Gulf, Gulf of Oman, east Asian marginal seas, and Adriatic Sea. The data include temperature, salinity, bioluminescence, chlorophyll-a fluorescence, transmissivity at two different wavelengths (TRed at 670 nm, TBlue at 490 nm), and back scattering coefficient (bRed at 700 nm, bBlue at 470 nm). Transfer and correlation functions between the hydrographic and optical parameters are obtained. Bioluminescence and fluorescence maxima, transmissivity minimum with their corresponding depths, red and blue laser beam peak attenuation coefficients are identified from the optical profiles. Evident correlations are found between the ocean mixed layer depth and the blue and red laser beam peak attenuation coefficients, bioluminescence and fluorescence maxima in the Adriatic Sea, Arabian Gulf, Gulf of Oman, and Philippine Sea. Based on the observational data, an effective algorithm is recommended for solving the radiative transfer equation (RTE) for predicting underwater laser radiance.

Multi-spectral wide-field imaging for PplX PDT dosimetry of skin (Conference Presentation)

NASA Astrophysics Data System (ADS)

LaRochelle, Ethan; Chun, Hayden H.; Hasan, Tayyaba; Pogue, Brian W.; Maytin, Edward V.; Chapman, Michael S.; Davis, Scott C.

2016-03-01

Actinic Kertoses (AK) are common pre-cancerous lesions associated with sun-damaged skin. While generally benign, the condition can progress to squamous cell carcinoma (SCC) and is a particular concern for immunosuppressed patients who are susceptible to uncontrolled AK and SCC. Among the FDA-approved treatment options for AK, ALA-based photodynamic therapy is unique in that it is non-scarring and can be repeated on the same area. However, response rates vary widely due to variations in drug and light delivery, PpIX production, and tissue oxygenation. Thus, developing modalities to predict response is critical to enable patient-specific treatment-enhancing interventions. To that end, we have developed a wide-field spectrally-resolved fluorescence imaging system capable of red and blue light excitation. While blue light excites PpIX efficiently, poor photon penetration limits the image content to superficial layers of skin. Red light excitation, on the other hand, can reveal fluorescence information originating from deeper in tissue, which may provide relevant information about PpIX distribution. Our instrument illuminates the skin via a fiber-based ring illuminator, into which is coupled sequentially a white light source, and blue and red laser diodes. Light emitted from the tissue passes through a high-speed filter wheel with filters selected to resolve the PpIX emission spectrum. This configuration enables the use of spectral fitting to decouple PpIX fluorescence from background signal, improving sensitivity to low concentrations of PpIX. Images of tissue-simulating phantoms and animal models confirm a linear response to PpIX, and the ability to image sub-surface PpIX inaccessible with blue light using red excitation.

The First Mutant of the Aequorea victoria Green Fluorescent Protein That Forms a Red Chromophore†

PubMed Central

Mishin, Alexander S.; Subach, Fedor V.; Yampolsky, Ilia V.; King, William; Lukyanov, Konstantin A.; Verkhusha, Vladislav V.

2010-01-01

Green fluorescent protein (GFP) from a jellyfish, Aequorea victoria, and its mutants are widely used in biomedical studies as fluorescent markers. In spite of the enormous efforts of academia and industry toward generating its red fluorescent mutants, no GFP variants with emission maximum at more than 529 nm have been developed during the 15 years since its cloning. Here, we used a new strategy of molecular evolution aimed at generating a red-emitting mutant of GFP. As a result, we have succeeded in producing the first GFP mutant that substantially matures to the red-emitting state with excitation and emission maxima at 555 and 585 nm, respectively. A novel, nonoxidative mechanism for formation of the red chromophore in this mutant that includes a dehydration of the Ser65 side chain has been proposed. Model experiments showed that the novel dual-color GFP mutant with green and red emission is suitable for multicolor flow cytometry as an additional color since it is clearly separable from both green and red fluorescent tags. PMID:18366185

The interaction of C.I. acid red 27 with human hemoglobin in solution.

PubMed

Wang, Yan-Qing; Zhang, Hong-Mei; Tang, Bo-Ping

2010-08-02

The nature of the interaction between human hemoglobin and C.I. acid red 27 was investigated systematically by ultraviolet-vis absorbance, circular dichroism, fluorescence, synchronous fluorescence, and three-dimensional fluorescence spectra techniques at pH 7.40. The quenching mechanism, binding constants, and the number of binding sites were determined by the quenching of human hemoglobin fluorescence in presence of C.I. acid red 27. The results showed that the nature of the quenching was of static type and the process of binding acid red 27 on human hemoglobin was a spontaneous molecular interaction procedure. The electrostatic and hydrophobic interactions played a major role in stabilizing the complex; The distance r between donor and acceptor was obtained to be 4.40 nm according to Förster's theory; The effect of acid red 27 on the conformation of human hemoglobin was analyzed using synchronous fluorescence, circular dichroism and three-dimensional fluorescence spectra. 2010 Elsevier B.V. All rights reserved.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wang, Shengyan; Shan, Liang; Fan, Yong

Three isomorphic lanthanide metal-organic frameworks (Ln-MOFs) [LnL(H{sub 2}O){sub 2}]·2H{sub 2}O (Ln=Tb for 1, Eu for 2, Gd for 3) have been constructed from flexible organic ligand 4-(2-carboxyphenoxy)benzene-1,3-dioic acid (H{sub 3}L). They exhibit two-dimensional (2D) layered structure with the rhombus windows along the b axis. This network can be described as a shubnikov plane net with Schäfli symbol of (4{sup 3}){sub 2}(4{sup 6}.6{sup 6}.8{sup 3}). Solid state luminescent studies indicate that 1 and 2 show the characteristic red, and green emissions of the corresponding Ln{sup 3+} ions, respectively, while 3 exhibits blue emission arising from the organic ligand. Then by adjustingmore » the relative amounts of different luminescent components into the well-defined host framework, a series of new co-doped Ln-MOF, Tb{sub 1−x}Eu{sub x}L (4) (x refers to the molar ratios of Eu{sup 3+} and Tb{sup 3+}), with tunable luminescence have been fabricated. The luminescent color of 4 can be tuned from green to red due to the energy transfer from the Tb{sup 3+} to Eu{sup 3+} ions by changing the doping concentration of the Eu{sup 3+} ions. In addition, 2 exhibits good stability in different solvents and excellent fluorescence sensing for small molecules, especially for CH{sub 3}CN and nitrobenzene. - Graphical abstract: A series of isomorphic 2D layered Ln-MOFs have been constructed from flexible tricarboxylic ligand, showing tunable luminescence and excellent fluorescence sensing for small molecules, respectively. - Highlights: • Three isomorphic 2D layered Ln-MOFs were constructed by flexible tricarboxylic acid. • A series of Eu{sup 3+}/Tb{sup 3+} doped Ln-MOF 4 were fabricated and showed tunable luminescence. • Ln-MOF 2 exhibited excellent fluorescence sensing for small molecules.« less

FluoroMyelin™ Red is a bright, photostable and non-toxic fluorescent stain for live imaging of myelin

PubMed Central

Monsma, Paula C.; Brown, Anthony

2012-01-01

FluoroMyelin™ Red is a commercially available water-soluble fluorescent dye that has selectivity for myelin. This dye is marketed for the visualization of myelin in brain cryosections, though it is also used widely to stain myelin in chemically fixed tissue. Here we have investigated the suitability of FluoroMyelin™ Red as a vital stain for live imaging of myelin in myelinating co-cultures of Schwann cells and dorsal root ganglion neurons. We show that addition of FluoroMyelin™ Red to the culture medium results in selective staining of myelin sheaths, with an optimal staining time of 2 hours, and has no apparent adverse effect on the neurons, their axons, or the myelinating cells at the light microscopic level. The fluorescence is bright and photostable, permitting long-term time-lapse imaging. After rinsing the cultures with medium lacking FluoroMyelin™ Red, the dye diffuses out of the myelin with a half life of about 130 minutes resulting in negligible fluorescence remaining after 18–24 hours. In addition, the large Stokes shift exhibited by FluoroMyelin™ Red makes it possible to readily distinguish it from popular and widely used green and red fluorescent probes such as GFP and mCherry. Thus FluoroMyelin™ Red is a useful reagent for live fluorescence imaging studies on myelinated axons. PMID:22743799

Conversion of red fluorescent protein into a bright blue probe.

PubMed

Subach, Oksana M; Gundorov, Illia S; Yoshimura, Masami; Subach, Fedor V; Zhang, Jinghang; Grüenwald, David; Souslova, Ekaterina A; Chudakov, Dmitriy M; Verkhusha, Vladislav V

2008-10-20

We used a red chromophore formation pathway, in which the anionic red chromophore is formed from the neutral blue intermediate, to suggest a rational design strategy to develop blue fluorescent proteins with a tyrosine-based chromophore. The strategy was applied to red fluorescent proteins of the different genetic backgrounds, such as TagRFP, mCherry, HcRed1, M355NA, and mKeima, which all were converted into blue probes. Further improvement of the blue variant of TagRFP by random mutagenesis resulted in an enhanced monomeric protein, mTagBFP, characterized by the substantially higher brightness, the faster chromophore maturation, and the higher pH stability than blue fluorescent proteins with a histidine in the chromophore. The detailed biochemical and photochemical analysis indicates that mTagBFP is the true monomeric protein tag for multicolor and lifetime imaging, as well as the outstanding donor for green fluorescent proteins in Förster resonance energy transfer applications.

Advances in Fluorescence Sensing Systems for the Remote Assessment of Nitrogen Supply in Field Corn

NASA Technical Reports Server (NTRS)

Corp, L. A.; Chappelle, E. W.; McMurtrey, J. E.; Daughtry, C. S. T.; Kim, M. S.

2000-01-01

The studies described herein were conducted to better define changes in fluorescence properties of leaves from field grown corn (Zea mays L.) as they relate to varying levels of nitrogen (N) fertilization. This research was directed toward: 1) providing a remote non-destructive sensing technique to aid in the determination of optimal rates of N fertilization in corn crops and, 2) defining parameters for further development of fluorescence instrumentation to be operated remotely at field canopy levels. Fluorescence imaging bands centered in the blue (450 nm), green (525 nm), red (680 nm), and far-red (740 nm) and ratios of these bands were compared with the following plant parameters: rates of photosynthesis, N:C ratio, pigment concentrations, and grain yields. Both the fluorescence and physiological measures exhibited similar curvilinear responses to N fertilization level while significant linear correlations were obtained among fluorescence bands and band ratios to certain physiological measures of plant productivity. The red / blue, red / green, far-red / blue, far-red /green fluorescence ratios are well suited for remote observation and provided high correlations to grain yield, LAI, N:C, and chlorophyll contents. The results from this investigation indicate that fluorescence technology could aid in the determination of N fertilization requirements for corn. This discussion will also address design concepts and preliminary field trials of a mobile field-based Laser Induced Fluorescence Imaging System (LIFIS) capable of simultaneously acquiring images of four fluorescence emission bands from areas of plant canopies equaling 1 sq m and greater without interference of ambient solar radiation.

Sensitive spectroscopic detection of large and denatured protein aggregates in solution by use of the fluorescent dye Nile red.

PubMed

Sutter, Marc; Oliveira, Sabrina; Sanders, Niek N; Lucas, Bart; van Hoek, Arie; Hink, Mark A; Visser, Antonie J W G; De Smedt, Stefaan C; Hennink, Wim E; Jiskoot, Wim

2007-03-01

The fluorescent dye Nile red was used as a probe for the sensitive detection of large, denatured aggregates of the model protein beta-galactosidase (E. coli) in solution. Aggregates were formed by irreversible heat denaturation of beta-galactosidase below and above the protein's unfolding temperature of 57.4 degrees C, and the presence of aggregates in heated solutions was confirmed by static light scattering. Interaction of Nile red with beta-galactosidase aggregates led to a shift of the emission maximum (lambda (max)) from 660 to 611 nm, and to an increase of fluorescence intensity. Time-resolved fluorescence and fluorescence correlation spectroscopy (FCS) measurements showed that Nile red detected large aggregates with hydrodynamic radii around 130 nm. By steady-state fluorescence measurements, it was possible to detect 1 nM of denatured and aggregated beta-galactosidase in solution. The comparison with size exclusion chromatography (SEC) showed that native beta-galactosidase and small aggregates thereof had no substantial effect on the fluorescence of Nile red. Large aggregates were not detected by SEC, because they were excluded from the column. The results with beta-galactosidase demonstrate the potential of Nile red for developing complementary analytical methods that overcome the size limitations of SEC, and can detect the formation of large protein aggregates at early stages.

Detection limits of 405 nm and 633 nm excited PpIX fluorescence for brain tumor detection during stereotactic biopsy

NASA Astrophysics Data System (ADS)

Markwardt, Niklas; Götz, Marcus; Haj-Hosseini, Neda; Hollnburger, Bastian; Sroka, Ronald; Stepp, Herbert; Zelenkov, Petr; Rühm, Adrian

2016-04-01

5-aminolevulinic-acid-(5-ALA)-induced protoporphyrin IX (PpIX) fluorescence may be used to improve stereotactic brain tumor biopsies. In this study, the sensitivity of PpIX-based tumor detection has been investigated for two potential excitation wavelengths (405 nm, 633 nm). Using a 200 μm fiber in contact with semi-infinite optical phantoms containing ink and Lipovenös, PpIX detection limits of 4.0 nM and 200 nM (relating to 1 mW excitation power) were determined for 405 nm and 633 nm excitation, respectively. Hence, typical PpIX concentrations in glioblastomas of a few μM should be well detectable with both wavelengths. Additionally, blood layers of selected thicknesses were placed between fiber and phantom. Red excitation was shown to be considerably less affected by blood interference: A 50 μm blood layer, for instance, blocked the 405- nm-excited fluorescence completely, but reduced the 633-nm-excited signal by less than 50%. Ray tracing simulations demonstrated that - without blood layer - the sensitivity advantage of 405 nm rises for decreasing fluorescent volume from 50-fold to a maximum of 100-fold. However, at a tumor volume of 1 mm3, which is a typical biopsy sample size, the 633-nm-excited fluorescence signal is only reduced by about 10%. Further simulations revealed that with increasing fiber-tumor distance, the signal drops faster for 405 nm. This reduces the risk of detecting tumor tissue outside the needle's coverage, but diminishes the overlap between optically and mechanically sampled volumes. While 405 nm generally offers a higher sensitivity, 633 nm is more sensitive to distant tumors and considerably superior in case of blood-covered tumor tissue.

An optical marker based on the UV-induced green-to-red photoconversion of a fluorescent protein

PubMed Central

Ando, Ryoko; Hama, Hiroshi; Yamamoto-Hino, Miki; Mizuno, Hideaki; Miyawaki, Atsushi

2002-01-01

We have cloned a gene encoding a fluorescent protein from a stony coral, Trachyphyllia geoffroyi, which emits green, yellow, and red light. The protein, named Kaede, includes a tripeptide, His-Tyr-Gly, that acts as a green chromophore that can be converted to red. The red fluorescence is comparable in intensity to the green and is stable under usual aerobic conditions. We found that the green-red conversion is highly sensitive to irradiation with UV or violet light (350–400 nm), which excites the protonated form of the chromophore. The excitation lights used to elicit red and green fluorescence do not induce photoconversion. Under a conventional epifluorescence microscope, Kaede protein expressed in HeLa cells turned red in a graded fashion in response to UV illumination; maximal illumination resulted in a 2,000-fold increase in the ratio of red-to-green signal. These color-changing properties provide a simple and powerful technique for regional optical marking. A focused UV pulse creates an instantaneous plane source of red Kaede within the cytosol. The red spot spreads rapidly throughout the cytosol, indicating its free diffusibility in the compartment. The extensive diffusion allows us to delineate a single neuron in a dense culture, where processes originating from many different somata are present. Illumination of a focused UV pulse onto the soma of a Kaede-expressing neuron resulted in filling of all processes with red fluorescence, allowing visualization of contact sites between the red and green neurons of interest. PMID:12271129

Monomeric fluorescent timers that change color from blue to red report on cellular trafficking.

PubMed

Subach, Fedor V; Subach, Oksana M; Gundorov, Illia S; Morozova, Kateryna S; Piatkevich, Kiryl D; Cuervo, Ana Maria; Verkhusha, Vladislav V

2009-02-01

Based on the mechanism for chromophore formation in red fluorescent proteins, we developed three mCherry-derived monomeric variants, called fluorescent timers (FTs), that change their fluorescence from the blue to red over time. These variants exhibit distinctive fast, medium and slow blue-to-red chromophore maturation rates that depend on the temperature. At 37 degrees C, the maxima of the blue fluorescence are observed at 0.25, 1.2 and 9.8 h for the purified fast-FT, medium-FT and slow-FT, respectively. The half-maxima of the red fluorescence are reached at 7.1, 3.9 and 28 h, respectively. The FTs show similar timing behavior in bacteria, insect and mammalian cells. Medium-FT allowed for tracking of the intracellular dynamics of the lysosome-associated membrane protein type 2A (LAMP-2A) and determination of its age in the targeted compartments. The results indicate that LAMP-2A transport through the plasma membrane and early or recycling endosomes to lysosomes is a major pathway for LAMP-2A trafficking.

Enhancement of growth and lipid production from microalgae using fluorescent paint under the solar radiation.

PubMed

Seo, Yeong Hwan; Cho, Changsoon; Lee, Jung-Yong; Han, Jong-In

2014-12-01

Solar radiation has intensity that is too high to inhibit microalgae activity and is composed of wide light spectrum including ultraviolet (UV) range which cannot be utilized for microalgae. For these reasons, the modification of solar radiation is required for effective microalgae cultivation, and to do that, fluorescent paint was used for not only blocking excessive solar energy but also converting UV to visible light. With fluorescent aqueous layer, microalgae was protected from photoinhibition and could grow well, but there was difference in growth and lipid accumulation efficiencies depending on the color; maximum dry weight of 1.7 g/L was achieved in red paint, whereas best lipid content of 30% was obtained in blue one. This phenomenon was due to the different light spectrum made by colors. With simple process using fluorescent paint, modification of light was successfully done and allowing microalgae to grow under strong radiation such as solar radiation. Copyright © 2014 Elsevier Ltd. All rights reserved.

Protoporphyrin-IX fluorescence guided surgical resection in high-grade gliomas: The potential impact of human colour perception.

PubMed

Petterssen, Max; Eljamel, Sarah; Eljamel, Sam

2014-09-01

Protoporphyrin-IX (Pp-IX) fluorescence had been used frequently in recent years to guide microsurgical resection of high-grade gliomas (HGG), particularly following the publication of a randomized controlled trial demonstrating its advantages. However, Pp-IX fluorescence is dependent upon the surgeons' eyes' perception of red fluorescent colour. This study was designed to evaluate human eye fluorescence perception and establish a fluorescence scale. 20 of 108 pre-recorded images from intraoperative fluorescence of HGG were used to construct an 8-panel visual analogue fluorescence scale. The scale was validated by testing 56 participants with normal colour vision and three red-green colour-blind participants. For intra-rater agreement ten participants were tested twice and for inter-observer reliability the whole cohort were tested. The intra- and inter-observer reliability of the scale in normal colour vision participants was excellent. The scale was less reliable in the violet-blue panels of the scale. Colour-blind participants were not able to distinguish between red fluorescence and blue-violet colours. The 8-panel fluorescence scale is valid in differentiating red, pink and blue colours in a fluorescence surgical field among participants with normal colour perception and potentially useful to standardize fluorescence-guided surgery. However, colourblind surgeons should not use fluorescence-guided surgery. Copyright © 2014 Elsevier B.V. All rights reserved.

[Principles of changes of structural organization of cell membranes and functional properties of erythrocytes in neurotic disorders].

PubMed

Riazantseva, N V; Novitskiĭ, V V

2003-02-01

Investigation into structural, metabolic, and functional conditions of red blood cells was performed in 24 patients with a neurosis (neurasthenia, disturbance of asaptation) with the aid of electrophoretic division of proteins of the erythrocyte membrane, thin-layer chromatography, fluorescent probing of membranes, evaluation of peroxidative oxidation process, scanning and transmission electron microscopy, laser diphractometry, photometry. The patients with neurotic disorders at the early period after the influence of psychogenic factors (up to 3 months) revealed disorganization of lipid and protein composition of the red cell membrane, increase in microviscosity of its lipid phase, impairment of surface architectonics and ultrastructure of red cells, decrease of a deformation ability and increase of aggregate properties of erythrocytes. The authors treat stability of erythrocytes' homeostasis under the long-term influence of psychogenic factors from a viewpoint of adaptive changes in organism under the influence of neurogenic factors.

Spectrally resolved visualization of fluorescent dyes permeating into skin

NASA Astrophysics Data System (ADS)

Maeder, Ulf; Bergmann, Thorsten; Beer, Sebastian; Burg, Jan Michael; Schmidts, Thomas; Runkel, Frank; Fiebich, Martin

2012-03-01

We present a spectrally resolved confocal imaging approach to qualitatively asses the overall uptake and the penetration depth of fluorescent dyes into biological tissue. We use a confocal microscope with a spectral resolution of 5 nm to measure porcine skin tissue after performing a Franz-Diffusion experiment with a submicron emulsion enriched with the fluorescent dye Nile Red. The evaluation uses linear unmixing of the dye and the tissue autofluorescence spectra. The results are combined with a manual segmentation of the skin's epidermis and dermis layers to assess the penetration behavior additionally to the overall uptake. The diffusion experiments, performed for 3h and 24h, show a 3-fold increased dye uptake in the epidermis and dermis for the 24h samples. As the method is based on spectral information it does not face the problem of superimposed dye and tissue spectra and therefore is more precise compared to intensity based evaluation methods.

Polyhydroxyalkanoate (PHA) Granules Have no Phospholipids.

PubMed

Bresan, Stephanie; Sznajder, Anna; Hauf, Waldemar; Forchhammer, Karl; Pfeiffer, Daniel; Jendrossek, Dieter

2016-05-25

Polyhydroxybutyrate (PHB) granules, also designated as carbonosomes, are supra-molecular complexes in prokaryotes consisting of a PHB polymer core and a surface layer of structural and functional proteins. The presence of suspected phospholipids in the surface layer is based on in vitro data of isolated PHB granules and is often shown in cartoons of the PHB granule structure in reviews on PHB metabolism. However, the in vivo presence of a phospholipid layer has never been demonstrated. We addressed this topic by the expression of fusion proteins of DsRed2EC and other fluorescent proteins with the phospholipid-binding domain (LactC2) of lactadherin in three model organisms. The fusion proteins specifically localized at the cell membrane of Ralstonia eutropha but did not co-localize with PHB granules. The same result was obtained for Pseudomonas putida, a species that accumulates another type of polyhydroxyalkanoate (PHA) granules related to PHB. Notably, DsRed2EC-LactC2 expressed in Magnetospirillum gryphiswaldense was detected at the position of membrane-enclosed magnetosome chains and at the cytoplasmic membrane but not at PHB granules. In conclusion, the carbonosomes of representatives of α-proteobacteria, β-proteobacteria and γ-proteobacteria have no phospholipids in vivo and we postulate that the PHB/PHA granule surface layers in natural producers generally are free of phospholipids and consist of proteins only.

Using Green and Red Fluorescent Proteins to Teach Protein Expression, Purification, and Crystallization

ERIC Educational Resources Information Center

Wu, Yifeng; Zhou, Yangbin; Song, Jiaping; Hu, Xiaojian; Ding, Yu; Zhang, Zhihong

2008-01-01

We have designed a laboratory curriculum using the green and red fluorescent proteins (GFP and RFP) to visualize the cloning, expression, chromatography purification, crystallization, and protease-cleavage experiments of protein science. The EGFP and DsRed monomer (mDsRed)-coding sequences were amplified by PCR and cloned into pMAL (MBP-EGFP) or…

Frequency domain fluorescent diffuse tomography of small animals with DsRed2-expressed tumors

NASA Astrophysics Data System (ADS)

Turchin, Ilya V.; Savitsky, Alexander P.; Kamensky, Vladislav A.; Plehanov, Vladimir I.; Orlova, Anna G.; Sergeeva, Ekaterina A.; Kleshnin, Mikhail S.; Shirmanova, Marina V.

2006-02-01

The main applications of fluorescent proteins (FPs) are monitoring tumor growth, angiogenesis, metastases formation and effects of new classes of drugs. Different types of tomography allow fluorescence imaging of tumors located deep in human or animal tissue. These techniques were used for investigation of the distribution of near-infrared fluorescent probes, but only a few works are devoted to fluorescence tomography in visible light. In this work, preliminary results of the frequency domain fluorescent diffuse tomography (FD FDT) method in application to DsRed2 protein as a fluorescent agent are presented. For the first step of our experiments we utilized second harmonic generation of Nd:YAG laser (532 nm) modulated by low frequency (1 kHz) in the experimental setup. The transilluminative planar configuration was used in the setup. A series of model experiments has been conducted and show good agreement between theoretical and experimental fluorescence intensity. Post mortem experiments with capsules containing DsRed2 and scattering solution introduced into esophagus of rats to simulate tumor formation have been conducted. The results of these experiments show that sensitivity of the setup is sufficient to detect DsRed2 in concentrations similar to those in FP-expressed tumor, but the contrast is not enough high to separate fluorescence of DsRed2 and surrounding tissues. The setup can be significantly improved by utilizing high-frequency modulation (110 MHz using acousto-optical modulator) of the excitation light and precise phase measurements due to difference in fluorescence life-time of FPs and surrounding tissues. An algorithm of processing a fluorescent image based on calculating zero of maximum curvature was employed for detection of fluorescent inclusions boundaries in the image.

Quantitative description of charge-carrier transport in a white organic light-emitting diode

NASA Astrophysics Data System (ADS)

Schober, M.; Anderson, M.; Thomschke, M.; Widmer, J.; Furno, M.; Scholz, R.; Lüssem, B.; Leo, K.

2011-10-01

We present a simulation model for the analysis of charge-carrier transport in organic thin-film devices, and apply it to a three-color white hybrid organic light-emitting diode (OLED) with fluorescent blue and phosphorescent red and green emission. We simulate a series of single-carrier devices, which reconstruct the OLED layer sequence step by step. Thereby, we determine the energy profiles for hole and electron transport, show how to discern bulk from interface limitation, and identify trap states.

Raman scattering and red fluorescence in the photochemical transformation of dry tryptophan particles

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lai, Chih Wei; Schwab, Mark; Hill, Steven C.

Tryptophan is a fluorescent amino acid common in proteins. Its absorption is largest for wavelengths λ ≲ 290 nm and its fluorescence emissions peak around 300–350 nm, depending upon the local environment. Here we report the observation of red fluorescence near 600 nm emerging from 488-nm continuous-wave (CW) laser photoexcitation of dry tryptophan (Trp) particles. With an excitation intensity below 0.5 kW/cm 2, dry Trp particles yield distinctive Raman scattering peaks in the presence of relatively weak and spectrally broad emissions with λ ~500–700 nm, allowing estimation of particle temperature at low excitation intensities. When the photoexcitation intensity is increasedmore » to 1 kW/cm 2 or more for a few minutes, fluorescence intensity dramatically increases by more than two orders of magnitude. The fluorescence continues to increase in intensity and gradually shift to the red when photoexcitation intensity and the duration of exposure are increased. The resulting products absorb at visible wavelengths and generate red fluorescence with λ ~ 650–800 nm with 633-nm CW laser excitation. In conclusion, we attribute the emergence of orange and red fluorescence in the Trp products to a photochemical transformation that is instigated by weak optical transitions to triplet states in Trp with 488-nm excitation and which may be expedited by a photothermal effect.« less

Raman scattering and red fluorescence in the photochemical transformation of dry tryptophan particles

DOE PAGES

Lai, Chih Wei; Schwab, Mark; Hill, Steven C.; ...

2016-05-19

Tryptophan is a fluorescent amino acid common in proteins. Its absorption is largest for wavelengths λ ≲ 290 nm and its fluorescence emissions peak around 300–350 nm, depending upon the local environment. Here we report the observation of red fluorescence near 600 nm emerging from 488-nm continuous-wave (CW) laser photoexcitation of dry tryptophan (Trp) particles. With an excitation intensity below 0.5 kW/cm 2, dry Trp particles yield distinctive Raman scattering peaks in the presence of relatively weak and spectrally broad emissions with λ ~500–700 nm, allowing estimation of particle temperature at low excitation intensities. When the photoexcitation intensity is increasedmore » to 1 kW/cm 2 or more for a few minutes, fluorescence intensity dramatically increases by more than two orders of magnitude. The fluorescence continues to increase in intensity and gradually shift to the red when photoexcitation intensity and the duration of exposure are increased. The resulting products absorb at visible wavelengths and generate red fluorescence with λ ~ 650–800 nm with 633-nm CW laser excitation. In conclusion, we attribute the emergence of orange and red fluorescence in the Trp products to a photochemical transformation that is instigated by weak optical transitions to triplet states in Trp with 488-nm excitation and which may be expedited by a photothermal effect.« less

Raman scattering and red fluorescence in the photochemical transformation of dry tryptophan particles.

PubMed

Lai, Chih Wei; Schwab, Mark; Hill, Steven C; Santarpia, Joshua; Pan, Yong-Le

2016-05-30

Tryptophan is a fluorescent amino acid common in proteins. Its absorption is largest for wavelengths λ ≲ 290 nm and its fluorescence emissions peak around 300-350 nm, depending upon the local environment. Here we report the observation of red fluorescence near 600 nm emerging from 488-nm continuous-wave (CW) laser photoexcitation of dry tryptophan (Trp) particles. With an excitation intensity below 0.5 kW/cm², dry Trp particles yield distinctive Raman scattering peaks in the presence of relatively weak and spectrally broad emissions with λ ∼500-700 nm, allowing estimation of particle temperature at low excitation intensities. When the photoexcitation intensity is increased to 1 kW/cm² or more for a few minutes, fluorescence intensity dramatically increases by more than two orders of magnitude. The fluorescence continues to increase in intensity and gradually shift to the red when photoexcitation intensity and the duration of exposure are increased. The resulting products absorb at visible wavelengths and generate red fluorescence with λ ∼ 650-800 nm with 633-nm CW laser excitation. We attribute the emergence of orange and red fluorescence in the Trp products to a photochemical transformation that is instigated by weak optical transitions to triplet states in Trp with 488-nm excitation and which may be expedited by a photothermal effect.

Multispectral scanning laser ophthalmoscopy combined with optical coherence tomography for simultaneous in vivo mouse retinal imaging

NASA Astrophysics Data System (ADS)

Zhang, Pengfei; Zam, Azhar; Jian, Yifan; Wang, Xinlei; Burns, Marie E.; Sarunic, Marinko V.; Pugh, Edward N.; Zawadzki, Robert J.

2015-03-01

A compact, non-invasive multi-modal system has been developed for in vivo mouse retina imaging. It is configured for simultaneously detecting green and red fluorescent protein signals with scanning laser ophthalmoscopy (SLO) back-scattered light from the SLO illumination beam, and depth information about different retinal layers by means of Optical Coherence Tomography (OCT). Simultaneous assessment of retinal characteristics with different modalities can provide a wealth of information about the structural and functional changes in the retinal neural tissue and chorio-retinal vasculature in vivo. Additionally, simultaneous acquisition of multiple channels facilitates analysis of the data of different modalities by automatic temporal and structural co-registration. As an example of the instrument's performance we imaged the retina of a mouse with constitutive expression of GFP in microglia cells (Cx3cr1GFP/+), and which also expressed the red fluorescent protein mCherry in Müller glial cells by means of adeno-associated virus delivery (AAV2) of an mCherry cDNA driven by the GFAP (glial fibrillary acid protein) promoter.

Deep-red to near-infrared fluorescent dyes: Synthesis, photophysical properties, and application in cell imaging

NASA Astrophysics Data System (ADS)

Li, Qi; Liu, Weimin; Wu, Jiasheng; Zhou, Bingjiang; Niu, Guangle; Zhang, Hongyan; Ge, Jiechao; Wang, Pengfei

2016-07-01

More and more attention has been paid to the design of new fluorescent imaging agents with good photostability and water solubility, especially those with emissions in the deep-red and near-infrared regions. In this work, we designed and synthesized four novel fluorescent dyes with deep-red or NIR fluorescence by hybridizing coumarin and pyronin moieties based on our previous work. Introduction of carboxylic acid in the dyes not only imparted the dyes with water solubility but also provided a versatile sensing platform for designing the fluorescent probes and sensors of biomolecules. The photophysical properties of these new dyes were investigated through absorption and fluorescence spectroscopy. Cell imaging experiments showed that esterification products could selectively stain lysosomes with good photostability, thereby indicating that they could be useful in the development of fluorescent probes for bioimaging.

Efficient hybrid white organic light-emitting diodes for application of triplet harvesting with simple structure

NASA Astrophysics Data System (ADS)

Hwang, Kyo Min; Lee, Song Eun; Lee, Sungkyu; Yoo, Han Kyu; Baek, Hyun Jung; Kim, Young Kwan; Kim, Jwajin; Yoon, Seung Soo

2016-08-01

In this study, we fabricated hybrid white organic light-emitting diodes (WOLEDs) based on triplet harvesting with a simple structure. All the hole transporting material and host in the emitting layer (EML) of devices utilized the same material N,N'-di-1-naphthalenyl-N,N'-diphenyl [1,1':4',1″:4″,1‴-quaterphenyl]-4,4‴-diamine (4P-NPD), which is known to be blue fluorescent material. Simple hybrid WOLEDs were fabricated with blue fluorescent, green and red phosphorescent materials. We investigated the effect of triplet harvesting (TH) by an exciton generation zone on simple hybrid WOLEDs. The simple hybrid WOLEDs characteristically had a dominant hole mobility, so an exciton generation zone was expected in the EML. Additionally, the optimal the thickness of the hole transporting layer and electron transporting layer was fabricated a simple hybrid WOLEDs. The simple hybrid WOLED exhibits a maximum luminous efficiency of 29.3 cd/A and a maximum external quantum efficiency of 11.2%. The Commission Internationale de l'Éclairage (International Commission on Illumination) coordinates were (0.45, 0.43) at about 10,000 cd/m2.

HcRed, a Genetically Encoded Fluorescent Binary Cross-Linking Agent for Cross-Linking of Mitochondrial ATP Synthase in Saccharomyces cerevisiae

PubMed Central

Gong, Lan; Ramm, Georg; Devenish, Rodney J.; Prescott, Mark

2012-01-01

Genetically encoded fluorescent cross-linking agents represent powerful tools useful both for visualising and modulating protein interactions in living cells. The far-red fluorescent protein HcRed, which is fluorescent only in a dimer form, can be used to promote the homo-dimerisation of target proteins, and thereby yield useful information about biological processes. We have in yeast cells expressed HcRed fused to a subunit of mitochondrial ATP synthase (mtATPase). This resulted in cross-linking of the large multi-subunit mtATPase complex within the inner-membrane of the mitochondrion. Fluorescence microscopy revealed aberrant mitochondrial morphology, and mtATPase complexes isolated from mitochondria were recovered as fluorescent dimers under conditions where complexes from control mitochondria were recovered as monomers. When viewed by electron microscopy normal cristae were absent from mitochondria in cells in which mATPase complexes were cross-linked. mtATPase dimers are believed to be the building blocks that are assembled into supramolecular mtATPase ribbons that promote the formation of mitochondrial cristae. We propose that HcRed cross-links mATPase complexes in the mitochondrial membrane hindering the normal assembly/disassembly of the supramolecular forms of mtATPase. PMID:22496895

Is the red fluorescence of dental plaque related to its cariogenicity?

NASA Astrophysics Data System (ADS)

Bittar, Daniela G.; Pontes, Laura Regina A.; Calvo, Ana Flávia B.; Novaes, Tatiane F.; Braga, Mariana M.; Freitas, Patrícia M.; Tabchoury, Cinthia P. M.; Mendes, Fausto M.

2014-06-01

It has been speculated that the red fluorescence emitted by dental plaque could be related to its cariogenicity. To test this hypothesis, we designed this crossover in situ study, with two experimental phases of 14 days each. Seventeen volunteers, wearing a palatal appliance with bovine enamel blocks, were instructed to drip a 20% sucrose solution (experimental group) or purified water (control group) onto the enamel blocks eight times daily. The specimens were removed after 4, 7, 10, and 14 days, and the red fluorescence of dental plaque formed on the enamel blocks was assessed using a quantitative light-induced fluorescence device. After the plaque removal, surface and cross-sectional microhardness tests were performed to assess the mineral loss. The comparisons were made by a multilevel linear regression analysis. We observed a significant increase in the red fluorescence of the dental plaque after longer periods of formation, but this trend was verified in both groups. The mineral loss assessed by the microhardness techniques, contrariwise, showed a significant increase only in the experimental group. In conclusion, the red fluorescence emitted by the dental plaque indicates a mature biofilm, but this fact is not necessarily associated with its cariogenicity.

Red fluorescent protein responsible for pigmentation in trematode-infected Porites compressa tissues.

PubMed

Palmer, Caroline V; Roth, Melissa S; Gates, Ruth D

2009-02-01

Reports of coral disease have increased dramatically over the last decade; however, the biological mechanisms that corals utilize to limit infection and resist disease remain poorly understood. Compromised coral tissues often display non-normal pigmentation that potentially represents an inflammation-like response, although these pigments remain uncharacterized. Using spectral emission analysis and cryo-histological and electrophoretic techniques, we investigated the pink pigmentation associated with trematodiasis, infection with Podocotyloides stenometre larval trematode, in Porites compressa. Spectral emission analysis reveals that macroscopic areas of pink pigmentation fluoresce under blue light excitation (450 nm) and produce a broad emission peak at 590 nm (+/-6) with a 60-nm full width at half maximum. Electrophoretic protein separation of pigmented tissue extract confirms the red fluorescence to be a protein rather than a low-molecular-weight compound. Histological sections demonstrate green fluorescence in healthy coral tissue and red fluorescence in the trematodiasis-compromised tissue. The red fluorescent protein (FP) is limited to the epidermis, is not associated with cells or granules, and appears unstructured. These data collectively suggest that the red FP is produced and localized in tissue infected by larval trematodes and plays a role in the immune response in corals.

Monomeric red fluorescent proteins with a large Stokes shift.

PubMed

Piatkevich, Kiryl D; Hulit, James; Subach, Oksana M; Wu, Bin; Abdulla, Arian; Segall, Jeffrey E; Verkhusha, Vladislav V

2010-03-23

Two-photon microscopy has advanced fluorescence imaging of cellular processes in living animals. Fluorescent proteins in the blue-green wavelength range are widely used in two-photon microscopy; however, the use of red fluorescent proteins is limited by the low power output of Ti-Sapphire lasers above 1,000 nm. To overcome this limitation we have developed two red fluorescent proteins, LSS-mKate1 and LSS-mKate2, which possess large Stokes shifts with excitation/emission maxima at 463/624 and 460/605 nm, respectively. These LSS-mKates are characterized by high pH stability, photostability, rapid chromophore maturation, and monomeric behavior. They lack absorbance in the green region, providing an additional red color to the commonly used red fluorescent proteins. Substantial overlap between the two-photon excitation spectra of the LSS-mKates and blue-green fluorophores enables multicolor imaging using a single laser. We applied this approach to a mouse xenograft model of breast cancer to intravitally study the motility and Golgi-nucleus alignment of tumor cells as a function of their distance from blood vessels. Our data indicate that within 40 mum the breast cancer cells show significant polarization towards vessels in living mice.

Green Synthesis of Red-Emitting Carbon Nanodots as a Novel "Turn-on" Nanothermometer in Living Cells.

PubMed

Wang, Chuanxi; Jiang, Kaili; Wu, Qian; Wu, Jiapeng; Zhang, Chi

2016-10-04

Temperature measurements in biology and medical diagnostics, along with sensitive temperature probing of living cells, is of great importance; however, it still faces significant challenges. Herein, a novel "turn-on" carbon-dot-based fluorescent nanothermometry device for spatially resolved temperature measurements in living cells is presented. The carbon nanodots (CNDs) are prepared by a green microwave-assisted method and exhibit red fluorescence (λem =615 nm) with high quantum yields (15 %). Then, an on-off fluorescent probe is prepared for detecting glutathione (GSH) based on aggregation-induced fluorescence quenching. Interestingly, the quenched fluorescence could be recovered by increasing temperature and the CNDs-GSH mixture could behave as an off-on fluorescent probe for temperature. Thus, red-emitting CNDs can be utilized for "turn-on" fluorescent nanothermometry through the fluorescence quenching and recovery processes, respectively. We employ MC3T3-E1 cells as an example model to demonstrate the red-emitting CNDs can function as "non-contact" tools for the accurate measurement of temperature and its gradient inside a living cell. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

[Discrimination of Red Tide algae by fluorescence spectra and principle component analysis].

PubMed

Su, Rong-guo; Hu, Xu-peng; Zhang, Chuan-song; Wang, Xiu-lin

2007-07-01

Fluorescence discrimination technology for 11 species of the Red Tide algae at genus level was constructed by principle component analysis and non-negative least squares. Rayleigh and Raman scattering peaks of 3D fluorescence spectra were eliminated by Delaunay triangulation method. According to the results of Fisher linear discrimination, the first principle component score and the second component score of 3D fluorescence spectra were chosen as discriminant feature and the feature base was established. The 11 algae species were tested, and more than 85% samples were accurately determinated, especially for Prorocentrum donghaiense, Skeletonema costatum, Gymnodinium sp., which have frequently brought Red tide in the East China Sea. More than 95% samples were right discriminated. The results showed that the genus discriminant feature of 3D fluorescence spectra of Red Tide algae given by principle component analysis could work well.

Determination of trace aluminum by fluorescence quenching method based on catalysis of potassium chlorate oxidizing alizarin red

NASA Astrophysics Data System (ADS)

Shao-Qin, Lin; Xuan, Lin; Shi-Rong, Hu; Li-Qing, Zeng; Yan, Wang; Li, Chen; Jia-Ming, Liu; Long-Di, Li

2005-11-01

A new method for the determination of trace aluminum has been proposed. It is based on the fact that alizarin red can emit strong and stable fluorescence at 80 °C for 30 min and Al 3+ can effectively catalyze potassium chlorate oxidizing alizarin red to form non-fluorescence complex which cause the fluorescence quenching. The linear dynamic range of this method is 0.040-4.00 ng l -1 with a detection limit of 5.3 pg l -1. The regression equation can be expressed as Δ If = 8.731 + 21.73 c (ng l -1), with the correlation coefficient r = 0.9992 ( n = 6). This sensitive, rapid and accurate method has been applied to the determination of trace aluminum(III) in human hair and tea samples successfully. What is more, the mechanism of catalyzing potassium chlorate oxidizing alizarin red by the fluorescence quenching method is also discussed.

Determination of trace aluminum by fluorescence quenching method based on catalysis of potassium chlorate oxidizing alizarin red.

PubMed

Shao-Qin, Lin; Xuan, Lin; Shi-Rong, Hu; Li-Qing, Zeng; Yan, Wang; Li, Chen; Jia-Ming, Liu; Long-Di, Li

2005-11-01

A new method for the determination of trace aluminum has been proposed. It is based on the fact that alizarin red can emit strong and stable fluorescence at 80 degrees C for 30 min and Al(3+) can effectively catalyze potassium chlorate oxidizing alizarin red to form non-fluorescence complex which cause the fluorescence quenching. The linear dynamic range of this method is 0.040-4.00 ngl(-1) with a detection limit of 5.3 pgl(-1). The regression equation can be expressed as DeltaI(f)=8.731+21.73c(Al(3+)) (ngl(-1)), with the correlation coefficient r=0.9992 (n=6). This sensitive, rapid and accurate method has been applied to the determination of trace aluminum(III) in human hair and tea samples successfully. What is more, the mechanism of catalyzing potassium chlorate oxidizing alizarin red by the fluorescence quenching method is also discussed.

Ocular fundus auto-fluorescence observations at different wavelengths in patients with age-related macular degeneration and diabetic retinopathy.

PubMed

Hammer, Martin; Königsdörffer, Ekkehart; Liebermann, Christiane; Framme, Carsten; Schuch, Günter; Schweitzer, Dietrich; Strobel, Jürgen

2008-01-01

Post-translational protein modification by lipid peroxidation products or glycation is a feature of aging as well as pathologic processes in postmitotic cells at the ocular fundus exposed to an oxidative environment. The accumulation of modified proteins such as those found in lipofuscin and advanced glycation end products (AGEs) contribute greatly to the fundus auto-fluorescence. The distinct fluorescence spectra of lipofuscin and AGE enable their differentiation in multispectral fundus fluorescence imaging. A dual-centre consecutive case series of 78 pseudo-phacic patients is reported. Digital colour fundus photographs as well as auto-fluorescence images were taken from 33 patients with age related macular degeneration (AMD), 13 patients with diabetic retinopathy (RD), or from 32 cases without pathologic findings (controls). Fluorescence was excited at 475-515 nm or 476-604 nm and recorded in the emission bands 530-675 nm or 675-715 nm, respectively. Fluorescence images excited at 475-515 nm were taken by a colour CCD-camera (colour-fluorescence imaging) enabling the separate recording of green and red fluorescence. The ratio of green versus red fluorescence was calculated within a representative region of each image. The 530-675 nm auto-fluorescence in AMD patients was dominated by the red emission (green vs. red ratio, g/r = 0.861). In comparison, the fluorescence of the diabetics was green-shifted (g/r = 0.946; controls: g/r = 0.869). Atrophic areas (geographic atrophy, laser scars) showed massive hypo-fluorescence in both emission bands. Hyper-fluorescent drusen and exudates, unobtrusive in the colour fundus images as well as in the fluorescence images with emission >667 nm, showed an impressive green-shift in the colour-fluorescence image. Lipofuscin is the dominant fluorophore at long wavelengths (>675 nm or red channel of the colour fluorescence image). In the green spectral region, we found an additional emission of collagen and elastin (optic disc, sclera) as well as deposits in drusen and exudates. The green shift of the auto-fluorescence in RD may be a hint of increased AGE concentrations.

On the heterogeneity of fluorescence lifetime of room temperature ionic liquids: onset of a journey for exploring red emitting dyes.

PubMed

Ghosh, Anup; Chatterjee, Tanmay; Mandal, Prasun K

2012-06-25

An excitation and emission wavelength dependent non-exponential fluorescence decay behaviour of room temperature ionic liquids (RTILs) has been noted. Average fluorescence lifetimes have been found to vary by a factor of three or more. Red emitting dyes dissolved in RTILs are found to follow hitherto unobserved single exponential fluorescence decay behaviour.

Comparison of red autofluorescing plaque and disclosed plaque-a cross-sectional study.

PubMed

Volgenant, Catherine M C; Fernandez Y Mostajo, Mercedes; Rosema, Nanning A M; van der Weijden, Fridus A; Ten Cate, Jacob M; van der Veen, Monique H

2016-12-01

The aim of this cross-sectional study was to assess the correlation between dental plaque scores determined by the measurement of red autofluorescence or by visualization with a two-tone solution. Clinical photographs were used for this study. Overnight plaque from the anterior teeth of 48 participants was assessed for red fluorescence on photographs (taken with a QLF-camera) using a modified Quigley & Hein (mQH) index. A two-tone disclosing solution was applied. Total disclosed plaque was clinically assessed using the mQH index. In addition, total and blue disclosed plaque was scored on clinical photographs using the mQH index. A strong correlation was observed between the total disclosed plaque scored on photographs and the clinical scores (r = 0.70 at site level; r = 0.88 at subject level). The correlation between red fluorescent plaque and total plaque, as assessed on the photographs, was moderate to strong and significant (r = 0.50 at the site level; r = 0.70 at the subject level), with the total plaque scores consistently higher than the red fluorescent plaque scores. The correlation between red fluorescent plaque and blue disclosed plaque was weak to moderate and significant (r = 0.30 at the site level; r = 0.50 at the subject level). Plaque, as scored on white-light photographs, corresponds well with clinically assessed plaque. A weak to moderate correlation between red fluorescing plaque and total disclosed plaque or blue disclosed plaque was found. What at present is considered to be matured dental plaque, which appears blue following the application of a two-tone disclosing solution, is not in agreement with red fluorescent dental plaque assessment.

Graphitic Nitrogen Triggers Red Fluorescence in Carbon Dots.

PubMed

Holá, Kateřina; Sudolská, Mária; Kalytchuk, Sergii; Nachtigallová, Dana; Rogach, Andrey L; Otyepka, Michal; Zbořil, Radek

2017-12-26

Carbon dots (CDs) are a stable and highly biocompatible fluorescent material offering great application potential in cell labeling, optical imaging, LED diodes, and optoelectronic technologies. Because their emission wavelengths provide the best tissue penetration, red-emitting CDs are of particular interest for applications in biomedical technologies. Current synthetic strategies enabling red-shifted emission include increasing the CD particle size (sp 2 domain) by a proper synthetic strategy and tuning the surface chemistry of CDs with suitable functional groups (e.g., carboxyl). Here we present an elegant route for preparing full-color CDs with well-controllable fluorescence at blue, green, yellow, or red wavelengths. The two-step procedure involves the synthesis of a full-color-emitting mixture of CDs from citric acid and urea in formamide followed by separation of the individual fluorescent fractions by column chromatography based on differences in CD charge. Red-emitting CDs, which had the most negative charge, were separated as the last fraction. The trend in the separation, surface charge, and red-shift of photoluminescence was caused by increasing amount of graphitic nitrogen in the CD structure, as was clearly proved by XPS, FT-IR, Raman spectroscopy, and DFT calculations. Importantly, graphitic nitrogen generates midgap states within the HOMO-LUMO gap of the undoped systems, resulting in significantly red-shifted light absorption that in turn gives rise to fluorescence at the low-energy end of the visible spectrum. The presented findings identify graphitic nitrogen as another crucial factor that can red-shift the CD photoluminescence.

Red fluorescence and 3-12 micron emission in NGC 2023, HD 44179, M 82, and Lynds 1780

NASA Technical Reports Server (NTRS)

Ryter, Charles; Dhendecourt, Louis B.

1989-01-01

A red excess observed in the Red Rectangle (HD 44179), was attributed to a possible molecular fluorescence mechanism was discovered in NGC 2023 and analyzed in subsequent work in this and other nebulae. An unexpected red light excess was also noticed in a high latitude dark cloud L 1780. The fluorescence was attributed to hydrogenated amorphous carbon by Duley (1985), on the basis of laboratory work. Alternatively, transitions between electronic states of free polycyclic aromatic hydrocarbon molecules, by-passing the cascade along the vibrational states was considered. In L 1780, the red excess was related to the 12 micron emission detected by IRAS. A quantitative comparison of the intensity of the red fluorescence and that of the 3 to 12 micron features is thus warranted in helping assess the physical properties of large interstellar molecules. The red fluorescence radiation, F(R), appears as a bump on the spectra between 0.6 and 0.9 micron. Values were deduced from the spectra for HD 44179, and for the high latitude cloud L 1780. Corrections for the extinction, both interstellar and internal to the nebulae, were included. The 3 to 12 micron brightness, F(IR), was obtained through integration of the spectra for NGC 2023, and for HD 44179 after removal of a smooth continuum due to hot large grains. The values of the ratio of fluorescence flux to the infrared flux, F(R)/F(IR), are summarized. Red fluorescence and infrared radiation are two separate ways to access to the size of the molecules through observation, and it is rewarding that both approaches give similar results. These findings bring a striking coherence into the physical description of the particles, and add further support to the initial attribution of the infrared features to polycyclic aromatic hydrocarbons (PAHs).

The 2013 FLEX-US Airborne Campaign at the Parker Tract Loblolly Pine Plantation in North Carolina, USA

NASA Technical Reports Server (NTRS)

Middleton, Elizabeth M.; Rascher, Uwe; Corp, Lawrence A.; Huemmrich, K. Fred; Cook, Bruce D.; Noormets, Asko; Schickling, Anke; Pinto, Francisco; Alonso, Luis; Damm, Alexander;

Solid-phase single molecule biosensing using dual-color colocalization of fluorescent quantum dot nanoprobes

NASA Astrophysics Data System (ADS)

Liu, Jianbo; Yang, Xiaohai; Wang, Kemin; Wang, Qing; Liu, Wei; Wang, Dong

2013-10-01

The development of solid-phase surface-based single molecule imaging technology has attracted significant interest during the past decades. Here we demonstrate a sandwich hybridization method for highly sensitive detection of a single thrombin protein at a solid-phase surface based on the use of dual-color colocalization of fluorescent quantum dot (QD) nanoprobes. Green QD560-modified thrombin binding aptamer I (QD560-TBA I) were deposited on a positive poly(l-lysine) assembled layer, followed by bovine serum albumin blocking. It allowed the thrombin protein to mediate the binding of the easily detectable red QD650-modified thrombin binding aptamer II (QD650-TBA II) to the QD560-TBA I substrate. Thus, the presence of the target thrombin can be determined based on fluorescent colocalization measurements of the nanoassemblies, without target amplification or probe separation. The detection limit of this assay reached 0.8 pM. This fluorescent colocalization assay has enabled single molecule recognition in a separation-free detection format, and can serve as a sensitive biosensing platform that greatly suppresses the nonspecific adsorption false-positive signal. This method can be extended to other areas such as multiplexed immunoassay, single cell analysis, and real time biomolecule interaction studies.The development of solid-phase surface-based single molecule imaging technology has attracted significant interest during the past decades. Here we demonstrate a sandwich hybridization method for highly sensitive detection of a single thrombin protein at a solid-phase surface based on the use of dual-color colocalization of fluorescent quantum dot (QD) nanoprobes. Green QD560-modified thrombin binding aptamer I (QD560-TBA I) were deposited on a positive poly(l-lysine) assembled layer, followed by bovine serum albumin blocking. It allowed the thrombin protein to mediate the binding of the easily detectable red QD650-modified thrombin binding aptamer II (QD650-TBA II) to the QD560-TBA I substrate. Thus, the presence of the target thrombin can be determined based on fluorescent colocalization measurements of the nanoassemblies, without target amplification or probe separation. The detection limit of this assay reached 0.8 pM. This fluorescent colocalization assay has enabled single molecule recognition in a separation-free detection format, and can serve as a sensitive biosensing platform that greatly suppresses the nonspecific adsorption false-positive signal. This method can be extended to other areas such as multiplexed immunoassay, single cell analysis, and real time biomolecule interaction studies. Electronic supplementary information (ESI) available: Absorbance and fluorescence spectra of quantum dot nanoprobes, electrophoresis analysis, and experimental setup for fluorescence imaging with dual channels. See DOI: 10.1039/c3nr03291d

Directed molecular evolution to design advanced red fluorescent proteins.

PubMed

Subach, Fedor V; Piatkevich, Kiryl D; Verkhusha, Vladislav V

2011-11-29

Fluorescent proteins have become indispensable imaging tools for biomedical research. Continuing progress in fluorescence imaging, however, requires probes with additional colors and properties optimized for emerging techniques. Here we summarize strategies for development of red-shifted fluorescent proteins. We discuss possibilities for knowledge-based rational design based on the photochemistry of fluorescent proteins and the position of the chromophore in protein structure. We consider advances in library design by mutagenesis, protein expression systems and instrumentation for high-throughput screening that should yield improved fluorescent proteins for advanced imaging applications.

Photoprotective Energy Dissipation in Higher Plants Involves Alteration of the Excited State Energy of the Emitting Chlorophyll(s) in the Light Harvesting Antenna II (LHCII)*

PubMed Central

Johnson, Matthew P.; Ruban, Alexander V.

2009-01-01

Non-photochemical quenching (NPQ), a mechanism of energy dissipation in higher plants protects photosystem II (PSII) reaction centers from damage by excess light. NPQ involves a reduction in the chlorophyll excited state lifetime in the PSII harvesting antenna (LHCII) by a quencher. Yet, little is known about the effect of the quencher on chlorophyll excited state energy and dynamics. Application of picosecond time-resolved fluorescence spectroscopy demonstrated that NPQ involves a red-shift (60 ± 5 cm−1) and slight enhancement of the vibronic satellite of the main PSII lifetime component present in intact chloroplasts. Whereas this fluorescence red-shift was enhanced by the presence of zeaxanthin, it was not dependent upon it. The red-shifted fluorescence of intact chloroplasts in the NPQ state was accompanied by red-shifted chlorophyll a absorption. Nearly identical absorption and fluorescence changes were observed in isolated LHCII complexes quenched in a low detergent media, suggesting that the mechanism of quenching is the same in both systems. In both cases, the extent of the fluorescence red-shift was shown to correlate with the lifetime of a component. The alteration in the energy of the emitting chlorophyll(s) in intact chloroplasts and isolated LHCII was also accompanied by changes in lutein 1 observed in their 77K fluorescence excitation spectra. We suggest that the characteristic red-shifted fluorescence emission reflects an altered environment of the emitting chlorophyll(s) in LHCII brought about by their closer interaction with lutein 1 in the quenching locus. PMID:19567871

Photoprotective energy dissipation in higher plants involves alteration of the excited state energy of the emitting chlorophyll(s) in the light harvesting antenna II (LHCII).

PubMed

Johnson, Matthew P; Ruban, Alexander V

2009-08-28

Non-photochemical quenching (NPQ), a mechanism of energy dissipation in higher plants protects photosystem II (PSII) reaction centers from damage by excess light. NPQ involves a reduction in the chlorophyll excited state lifetime in the PSII harvesting antenna (LHCII) by a quencher. Yet, little is known about the effect of the quencher on chlorophyll excited state energy and dynamics. Application of picosecond time-resolved fluorescence spectroscopy demonstrated that NPQ involves a red-shift (60 +/- 5 cm(-1)) and slight enhancement of the vibronic satellite of the main PSII lifetime component present in intact chloroplasts. Whereas this fluorescence red-shift was enhanced by the presence of zeaxanthin, it was not dependent upon it. The red-shifted fluorescence of intact chloroplasts in the NPQ state was accompanied by red-shifted chlorophyll a absorption. Nearly identical absorption and fluorescence changes were observed in isolated LHCII complexes quenched in a low detergent media, suggesting that the mechanism of quenching is the same in both systems. In both cases, the extent of the fluorescence red-shift was shown to correlate with the lifetime of a component. The alteration in the energy of the emitting chlorophyll(s) in intact chloroplasts and isolated LHCII was also accompanied by changes in lutein 1 observed in their 77K fluorescence excitation spectra. We suggest that the characteristic red-shifted fluorescence emission reflects an altered environment of the emitting chlorophyll(s) in LHCII brought about by their closer interaction with lutein 1 in the quenching locus.

Red Fluorescent Line Emission from Hydrogen Molecules in Diffuse Molecular Clouds

NASA Technical Reports Server (NTRS)

Neufeld, David A.; Spaans, Marco

1996-01-01

We have modeled the fluorescent pumping of electronic and vibrational emissions of molecular hydrogen (H2) within diffuse molecular clouds that are illuminated by ultraviolet continuum radiation. Fluorescent line intensities are predicted for transitions at ultraviolet, infrared, and red visible wavelengths as functions of the gas density, the visual extinction through the cloud, and the intensity of the incident UV continuum radiation. The observed intensity in each fluorescent transition is roughly proportional to the integrated rate of H2 photodissociation along the line of sight. Although the most luminous fluorescent emissions detectable from ground-based observatories lie at near-infrared wavelengths, we argue that the lower sky brightness at visible wavelengths makes the red fluorescent transitions a particularly sensitive probe. Fabry-Perot spectrographs of the type that have been designed to observe very faint diffuse Ha emissions are soon expected to yield sensitivities that will be adequate to detect H2 vibrational emissions from molecular clouds that are exposed to ultraviolet radiation no stronger than the mean radiation field within the Galaxy. Observations of red H2 fluorescent emission together with cospatial 21 cm H I observations could serve as a valuable probe of the gas density in diffuse molecular clouds.

In-stem labelling allows visualization of DNA strand displacements by distinct fluorescent colour change.

PubMed

Barrois, Sebastian; Wagenknecht, Hans-Achim

2013-05-21

The combination of thiazole orange (TO) and thiazole red (TR) as an internal pair of fluorescent DNA base surrogates ("DNA traffic lights") allows us to follow at least two consecutive DNA strand displacements in real time through a distinct fluorescence colour change from green to red and vice versa.

Photooxidation of Amplex Red to resorufin: implications of exposing the Amplex Red assay to light

PubMed Central

Zhao, Baozhong; Summers, Fiona A.; Mason, Ronald P.

2012-01-01

The Amplex Red assay, a fluorescent assay for the detection of H2O2, relies on the reaction of H2O2 and colorless, nonfluorescent Amplex Red with a 1:1 stoichiometry to form colored, fluorescent resorufin, catalyzed by horseradish peroxidase (HRP). We have found that resorufin is artifactually formed when Amplex Red is exposed to light. In the absence of H2O2 and HRP, the absorption and fluorescence spectra of Amplex Red changed during exposure to ambient room light or instrumental excitation light, clearly indicating that the fluorescent product resorufin had formed. This photochemistry was initiated by trace amounts of resorufin that are present in Amplex Red stock solutions. ESR spin-trapping studies demonstrated that superoxide radical was an intermediate in this process. Oxygen consumption measurements further confirmed that superoxide and H2O2 were artifactually produced by the photooxidation of Amplex Red. The artifactual formation of resorufin was also significantly increased by the presence of superoxide dismutase or HRP. This photooxidation process will result in a less sensitive assay for H2O2 under ambient light exposure and potentially invalid measurements under high energy exposure such as UVA irradiation. In general, precautions should be taken to minimize exposure to light during measurement of oxidative stress with Amplex Red. PMID:22765927

Photooxidation of Amplex Red to resorufin: implications of exposing the Amplex Red assay to light.

PubMed

Zhao, Baozhong; Summers, Fiona A; Mason, Ronald P

2012-09-01

The Amplex Red assay, a fluorescent assay for the detection of H(2)O(2), relies on the reaction of H(2)O(2) and colorless, nonfluorescent Amplex Red with a 1:1 stoichiometry to form colored, fluorescent resorufin, catalyzed by horseradish peroxidase (HRP). We have found that resorufin is artifactually formed when Amplex Red is exposed to light. In the absence of H(2)O(2) and HRP, the absorption and fluorescence spectra of Amplex Red changed during exposure to ambient room light or instrumental excitation light, clearly indicating that the fluorescent product resorufin had formed. This photochemistry was initiated by trace amounts of resorufin that are present in Amplex Red stock solutions. ESR spin-trapping studies demonstrated that superoxide radical was an intermediate in this process. Oxygen consumption measurements further confirmed that superoxide and H(2)O(2) were artifactually produced by the photooxidation of Amplex Red. The artifactual formation of resorufin was also significantly increased by the presence of superoxide dismutase or HRP. This photooxidation process will result in a less sensitive assay for H(2)O(2) under ambient light exposure and potentially invalid measurements under high energy exposure such as UVA irradiation. In general, precautions should be taken to minimize exposure to light during measurement of oxidative stress with Amplex Red. Published by Elsevier Inc.

[Fluorescence characterization of dissolved organic matter in the East China Sea after diatom red tide dispersion].

PubMed

Zhuo, Peng-ji; Zhao, Wei-hong

2009-05-01

Fluorescence excitation-emission spectroscopy (EEMS) was employed to analyze the 3-dimensional fluorescence of dissolved organic matter in the East China Sea after diatom red tide dispersion. The relationships between fluorescence peak intensity, and salinity and chlorophyll-a were discussed. The centers of protein-like fluorescence peaks dispersed at Exmax/Exmax = 270-280/290-315 nm (Peak B), 220-230/290-305 nm (Peak D), 230-240/335-350 nm (Peak S) and 280/320 nm (Peak T). Two humic-like peaks appeared at 255-270/435-480 nm (Peak A)and 330-350/420-480 nm (Peak C). High tyrosine-like intensity was observed in diatom red tide dispersion area, and tryptophan-like fluorescence was also found which was lower. High FIB/FIS showed that diatom red tide produced much tyrosine-like matter during dispersion. Peaks S, A and C had positive correlation with one another, and their distributions were similar, which decreased with distance increasing away from the shore. Good negative correlations between peaks S, A and C and salinity suggested that Jiangsu-Zhejiang coastal water was the same source of them. Correlations between fluorescence peak intensity and chlorophyll-a were not remarkable enough to clear the relationship between fluorescence and living algal matter. It was supposed that the living algal matter contributed little to the fluorescence intensity of algal dispersion seawater.

2D fluorescence spectroscopy for monitoring ion-exchange membrane based technologies - Reverse electrodialysis (RED).

PubMed

Pawlowski, Sylwin; Galinha, Claudia F; Crespo, João G; Velizarov, Svetlozar

2016-01-01

Reverse electrodialysis (RED) is one of the emerging, membrane-based technologies for harvesting salinity gradient energy. In RED process, fouling is an undesirable operation constraint since it leads to a decrease of the obtainable net power density due to increasing stack electric resistance and pressure drop. Therefore, early fouling detection is one of the main challenges for successful RED technology implementation. In the present study, two-dimensional (2D) fluorescence spectroscopy was used, for the first time, as a tool for fouling monitoring in RED. Fluorescence excitation-emission matrices (EEMs) of ion-exchange membrane surfaces and of natural aqueous streams were acquired during one month of a RED stack operation. Fouling evolvement on the ion-exchange membrane surfaces was successfully followed by 2D fluorescence spectroscopy and quantified using principal components analysis (PCA). Additionally, the efficiency of cleaning strategy was assessed by measuring the membrane fluorescence emission intensity before and after cleaning. The anion-exchange membrane (AEM) surface in contact with river water showed to be significantly affected due to fouling by humic compounds, which were found to cross through the membrane from the lower salinity (river water) to higher salinity (sea water) stream. The results obtained show that the combined approach of using 2D fluorescence spectroscopy and PCA has a high potential for studying fouling development and membrane cleaning efficiency in ion exchange membrane processes. Copyright © 2015 Elsevier Ltd. All rights reserved.

Suppression of Red Blood Cell Autofluorescence for Immunocytochemistry on Fixed Embryonic Mouse Tissue.

PubMed

Whittington, Niteace C; Wray, Susan

2017-10-23

Autofluorescence is a problem that interferes with immunofluorescent staining and complicates data analysis. Throughout the mouse embryo, red blood cells naturally fluoresce across multiple wavelengths, spanning the emission and excitation spectra of many commonly used fluorescent reporters, including antibodies, dyes, stains, probes, and transgenic proteins, making it difficult to distinguish assay fluorescence from endogenous fluorescence. Several tissue treatment methods have been developed to bypass this issue with varying degrees of success. Sudan Black B dye has been commonly used to quench autofluorescence, but can also introduce background fluorescence. Here we present a protocol for an alternative called TrueBlack Lipofuscin Autofluorescence Quencher. The protocol described in this unit demonstrates how TrueBlack efficiently quenches red blood cell autofluorescence across red and green wavelengths in fixed embryonic tissue without interfering with immunofluorescent signal intensity or introducing background staining. We also identify optimal incubation, concentration, and multiple usage conditions for routine immunofluorescence microscopy. © 2017 by John Wiley & Sons, Inc. Copyright © 2017 John Wiley & Sons, Inc.

A far-red fluorescent protein evolved from a cyanobacterial phycobiliprotein

PubMed Central

Rodriguez, Erik A.; Tran, Geraldine N.; Gross, Larry A.; Crisp, Jessica L.; Shu, Xiaokun; Lin, John Y.; Tsien, Roger Y.

2016-01-01

Far-red fluorescent proteins (FPs) are desirable for in vivo imaging because less light is scattered, absorbed, or reemitted by endogenous biomolecules. A new class of FP was developed from an allophycocyanin α-subunit (APCα). Native APC requires a lyase to incorporate phycocyanobilin. The evolved FP, named small Ultra-Red FP (smURFP), covalently attaches biliverdin (BV) without a lyase, and has 642/670 nm excitation/emission peaks, a large extinction coefficient (180,000 M−1cm−1) and quantum yield (18%), and comparable photostability to eGFP. SmURFP has significantly increased BV incorporation rate and protein stability compared to the bacteriophytochrome (BPH) FPs. BV supply is limited by membrane permeability, so expression of heme oxygenase-1 with heme precursors increases fluorescence of BPH/APCα FPs. SmURFP (but not BPH FPs) can incorporate a more membrane-permeant BV analog, making smURFP fluorescence in situ comparable to FPs from jellyfish/coral. A far-red/near-infrared fluorescent cell cycle indicator was created with smURFP and a BPH FP. PMID:27479328

Engineering of mCherry variants with long Stokes shift, red-shifted fluorescence, and low cytotoxicity

PubMed Central

Shen, Yi; Chen, Yingche; Wu, Jiahui; Shaner, Nathan C.; Campbell, Robert E.

2017-01-01

MCherry, the Discosoma sp. mushroom coral-derived monomeric red fluorescent protein (RFP), is a commonly used genetically encoded fluorophore for live cell fluorescence imaging. We have used a combination of protein design and directed evolution to develop mCherry variants with low cytotoxicity to Escherichia coli and altered excitation and emission profiles. These efforts ultimately led to a long Stokes shift (LSS)-mCherry variant (λex = 460 nm and λem = 610 nm) and a red-shifted (RDS)-mCherry variant (λex = 600 nm and λem = 630 nm). These new RFPs provide insight into the influence of the chromophore environment on mCherry’s fluorescence properties, and may serve as templates for the future development of fluorescent probes for live cell imaging. PMID:28241009

Novel multistep BRET-FRET energy transfer using nanoconjugates of firefly proteins, quantum dots, and red fluorescent proteins

NASA Astrophysics Data System (ADS)

Alam, Rabeka; Zylstra, Joshua; Fontaine, Danielle M.; Branchini, Bruce R.; Maye, Mathew M.

2013-05-01

Sequential bioluminescence resonance energy transfer (BRET) and fluorescence resonance energy transfer (FRET) from firefly luciferase to red fluorescent proteins using quantum dot or rod acceptor/donor linkers is described. The effect of morphology and tuned optical properties on the efficiency of this unique BRET-FRET system was evaluated.Sequential bioluminescence resonance energy transfer (BRET) and fluorescence resonance energy transfer (FRET) from firefly luciferase to red fluorescent proteins using quantum dot or rod acceptor/donor linkers is described. The effect of morphology and tuned optical properties on the efficiency of this unique BRET-FRET system was evaluated. Electronic supplementary information (ESI) available: Experimental details, Fig. S1 and Table S1-S4. See DOI: 10.1039/c3nr01842c

Polyhydroxyalkanoate (PHA) Granules Have no Phospholipids

PubMed Central

Bresan, Stephanie; Sznajder, Anna; Hauf, Waldemar; Forchhammer, Karl; Pfeiffer, Daniel; Jendrossek, Dieter

2016-01-01

Polyhydroxybutyrate (PHB) granules, also designated as carbonosomes, are supra-molecular complexes in prokaryotes consisting of a PHB polymer core and a surface layer of structural and functional proteins. The presence of suspected phospholipids in the surface layer is based on in vitro data of isolated PHB granules and is often shown in cartoons of the PHB granule structure in reviews on PHB metabolism. However, the in vivo presence of a phospholipid layer has never been demonstrated. We addressed this topic by the expression of fusion proteins of DsRed2EC and other fluorescent proteins with the phospholipid-binding domain (LactC2) of lactadherin in three model organisms. The fusion proteins specifically localized at the cell membrane of Ralstonia eutropha but did not co-localize with PHB granules. The same result was obtained for Pseudomonas putida, a species that accumulates another type of polyhydroxyalkanoate (PHA) granules related to PHB. Notably, DsRed2EC-LactC2 expressed in Magnetospirillum gryphiswaldense was detected at the position of membrane-enclosed magnetosome chains and at the cytoplasmic membrane but not at PHB granules. In conclusion, the carbonosomes of representatives of α-proteobacteria, β-proteobacteria and γ-proteobacteria have no phospholipids in vivo and we postulate that the PHB/PHA granule surface layers in natural producers generally are free of phospholipids and consist of proteins only. PMID:27222167

Antiphase dual-color correlation in a reactant-product pair imparts ultrasensitivity in reaction-linked double-photoswitching fluorescence imaging.

PubMed

Wan, Wei; Zhu, Ming-Qiang; Tian, Zhiyuan; Li, Alexander D Q

2015-04-08

A pair of reversible photochemical reactions correlates their reactant and product specifically, and such a correlation uniquely distinguishes their correlated signal from others that are not linked by this reversible reaction. Here a nanoparticle-shielded fluorophore is photodriven to undergo structural dynamics, alternating between a green-fluorescence state and a red-fluorescence state. As time elapses, the fluorophore can be in either state but not both at the same time. Thus, the red fluorescence is maximized while the green fluorescence is minimized and vice versa. Such an antiphase dual-color (AD) corelationship between the red and green fluorescence maxima as well as between their minima can be exploited to greatly improve the signal-to-noise ratio, thus enhancing the ultimate detection limit. Potential benefits of this correlation include elimination of all interferences originating from single-color dyes and signal amplification of AD photoswitching molecules by orders of magnitude.

Chemical reactivation of resin-embedded pHuji adds red for simultaneous two-color imaging with EGFP

PubMed Central

Guo, Wenyan; Liu, Xiuli; Liu, Yurong; Gang, Yadong; He, Xiaobin; Jia, Yao; Yin, Fangfang; Li, Pei; Huang, Fei; Zhou, Hongfu; Wang, Xiaojun; Gong, Hui; Luo, Qingming; Xu, Fuqiang; Zeng, Shaoqun

2017-01-01

The pH-sensitive fluorescent proteins enabling chemical reactivation in resin are useful tools for fluorescence microimaging. EGFP or EYFP is good for such applications. For simultaneous two-color imaging, a suitable red fluorescent protein is an urgent need. Here a pH-sensitive red fluorescent protein, pHuji, is selected and verified to remain pH-sensitive in HM20 resin. We observe 183% fluorescence intensity of pHuji in resin-embeded mouse brain and 29.08-fold fluorescence intensity of reactivated pHuji compared to the quenched state. pHuji and EGFP can be quenched and chemically reactivated simultaneously in resin, thus enabling simultaneous two-color micro-optical sectioning tomography of resin-embedded mouse brain. This method may greatly facilitate the visualization of neuronal morphology and neural circuits to promote understanding of the structure and function of the brain. PMID:28717566

Chemical reactivation of resin-embedded pHuji adds red for simultaneous two-color imaging with EGFP.

PubMed

Guo, Wenyan; Liu, Xiuli; Liu, Yurong; Gang, Yadong; He, Xiaobin; Jia, Yao; Yin, Fangfang; Li, Pei; Huang, Fei; Zhou, Hongfu; Wang, Xiaojun; Gong, Hui; Luo, Qingming; Xu, Fuqiang; Zeng, Shaoqun

2017-07-01

The pH-sensitive fluorescent proteins enabling chemical reactivation in resin are useful tools for fluorescence microimaging. EGFP or EYFP is good for such applications. For simultaneous two-color imaging, a suitable red fluorescent protein is an urgent need. Here a pH-sensitive red fluorescent protein, pHuji, is selected and verified to remain pH-sensitive in HM20 resin. We observe 183% fluorescence intensity of pHuji in resin-embeded mouse brain and 29.08-fold fluorescence intensity of reactivated pHuji compared to the quenched state. pHuji and EGFP can be quenched and chemically reactivated simultaneously in resin, thus enabling simultaneous two-color micro-optical sectioning tomography of resin-embedded mouse brain. This method may greatly facilitate the visualization of neuronal morphology and neural circuits to promote understanding of the structure and function of the brain.

UV red fluorescence of Eubacterium lentum.

PubMed Central

Mosca, A; Strong, C A; Finegold, S M

1993-01-01

Twenty-nine clinical isolates of Eubacterium lentum and two type species were evaluated for the ability to fluoresce under UV light. Twenty-one of the 29 isolates and both of the reference strains showed orange-to-red fluorescence. This fluorescence did not require blood or hemin in the culture media and did not fade upon air exposure. The fluorescent pigment, after extraction by 1 N NaOH, showed peak excitation at a wavelength of around 400 nm. The capacity of E. lentum to produce fluorescence may be a useful and time-sparing laboratory aid for its identification. PMID:8463378

Adapting photosynthesis to the near-infrared: non-covalent binding of phycocyanobilin provides an extreme spectral red-shift to phycobilisome core-membrane linker from Synechococcus sp. PCC7335.

PubMed

Miao, Dan; Ding, Wen-Long; Zhao, Bao-Qing; Lu, Lu; Xu, Qian-Zhao; Scheer, Hugo; Zhao, Kai-Hong

2016-06-01

Phycobiliproteins that bind bilins are organized as light-harvesting complexes, phycobilisomes, in cyanobacteria and red algae. The harvested light energy is funneled to reaction centers via two energy traps, allophycocyanin B and the core-membrane linker, ApcE1 (conventional ApcE). The covalently bound phycocyanobilin (PCB) of ApcE1 absorbs near 660 nm and fluoresces near 675 nm. In cyanobacteria capable of near infrared photoacclimation, such as Synechococcus sp. PCC7335, there exist even further spectrally red shifted components absorbing >700 nm and fluorescing >710 nm. We expressed the chromophore domain of the extra core-membrane linker from Synechococcus sp. PCC7335, ApcE2, in E. coli together with enzymes generating the chromophore, PCB. The resulting chromoproteins, PCB-ApcE2(1-273) and the more truncated PCB-ApcE2(24-245), absorb at 700 nm and fluoresce at 714 nm. The red shift of ~40 nm compared with canonical ApcE1 results from non-covalent binding of the chromophore by which its full conjugation length including the Δ3,3(1) double bond is preserved. The extreme spectral red-shift could not be ascribed to exciton coupling: dimeric PCB-ApcE2(1-273) and monomeric-ApcE2(24-245) absorbed and fluoresced similarly. Chromophorylation of ApcE2 with phycoerythrobilin- or phytochromobilin resulted in similar red shifts (absorption at 615 and 711 nm, fluorescence at 628 or 726 nm, respectively), compared to the covalently bound chromophores. The self-assembled non-covalent chromophorylation demonstrates a novel access to red and near-infrared emitting fluorophores. Brightly fluorescent biomarking was exemplified in E. coli by single-plasmid transformation. Copyright © 2016 Elsevier B.V. All rights reserved.

Far-red fluorescent probes for canonical and non-canonical nucleic acid structures: current progress and future implications.

PubMed

Suseela, Y V; Narayanaswamy, Nagarjun; Pratihar, Sumon; Govindaraju, Thimmaiah

2018-02-05

The structural diversity and functional relevance of nucleic acids (NAs), mainly deoxyribonucleic acid (DNA) and ribonucleic acid (RNA), are indispensable for almost all living organisms, with minute aberrations in their structure and function becoming causative factors in numerous human diseases. The standard structures of NAs, termed canonical structures, are supported by Watson-Crick hydrogen bonding. Under special physiological conditions, NAs adopt distinct spatial organisations, giving rise to non-canonical conformations supported by hydrogen bonding other than the Watson-Crick type; such non-canonical structures have a definite function in controlling gene expression and are considered as novel diagnostic and therapeutic targets. Development of molecular probes for these canonical and non-canonical DNA/RNA structures has been an active field of research. Among the numerous probes studied, probes with turn-on fluorescence in the far-red (600-750 nm) region are highly sought-after due to minimal autofluorescence and cellular damage. Far-red fluorescent probes are vital for real-time imaging of NAs in live cells as they provide good resolution and minimal perturbation of the cell under investigation. In this review, we present recent advances in the area of far-red fluorescent probes of DNA/RNA and non-canonical G-quadruplex structures. For the sake of continuity and completeness, we provide a brief overview of visible fluorescent probes. Utmost importance is given to design criteria, characteristic properties and biological applications, including in cellulo imaging, apart from critical discussion on limitations of the far-red fluorescent probes. Finally, we offer current and future prospects in targeting canonical and non-canonical NAs specific to cellular organelles, through sequence- and conformation-specific far-red fluorescent probes. We also cover their implications in chemical and molecular biology, with particular focus on decoding various disease mechanisms involving NAs.

Bioimaging of Fluorescence-Labeled Mitochondria in Subcutaneously Grafted Murine Melanoma Cells by the “In Vivo Cryotechnique”

PubMed Central

Lei, Ting; Huang, Zheng; Ohno, Nobuhiko; Wu, Bao; Sakoh, Takashi; Saitoh, Yurika; Saiki, Ikuo

2014-01-01

The microenvironments of organs with blood flow affect the metabolic profiles of cancer cells, which are influenced by mitochondrial functions. However, histopathological analyses of these aspects have been hampered by technical artifacts of conventional fixation and dehydration, including ischemia/anoxia. The purpose of this study was to combine the in vivo cryotechnique (IVCT) with fluorescent protein expression, and examine fluorescently labeled mitochondria in grafted melanoma tumors. The intensity of fluorescent proteins was maintained well in cultured B16-BL6 cells after cryotechniques followed by freeze-substitution (FS). In the subcutaneous tumors of mitochondria-targeted DsRed2 (mitoDsRed)-expressing cells, a higher number of cancer cells were found surrounding the widely opened blood vessels that contained numerous erythrocytes. Such blood vessels were immunostained positively for immunoglobulin M and ensheathed by basement membranes. MitoDsRed fluorescence was detected in scattering melanoma cells using the IVCT-FS method, and the total mitoDsRed volume in individual cancer cells was significantly decreased with the expression of markers of hypoxia. MitoDsRed was frequently distributed throughout the cytoplasm and in processes extending along basement membranes. IVCT combined with fluorescent protein expression is a useful tool to examine the behavior of fluorescently labeled cells and organelles. We propose that the mitochondrial volume is dynamically regulated in the hypoxic microenvironment and that mitochondrial distribution is modulated by cancer cell interactions with basement membranes. PMID:24394469

Do the fluorescent red eyes of the marine fish Tripterygion delaisi stand out? In situ and in vivo measurements at two depths.

PubMed

Harant, Ulrike K; Santon, Matteo; Bitton, Pierre-Paul; Wehrberger, Florian; Griessler, Thomas; Meadows, Melissa G; Champ, Connor M; Michiels, Nico K

2018-05-01

Since the discovery of red fluorescence in fish, much effort has been invested to elucidate its potential functions, one of them being signaling. This implies that the combination of red fluorescence and reflection should generate a visible contrast against the background. Here, we present in vivo iris radiance measurements of Tripterygion delaisi under natural light conditions at 5 and 20 m depth. We also measured substrate radiance of shaded and exposed foraging sites at those depths. To assess the visual contrast of the red iris against these substrates, we used the receptor noise model for chromatic contrasts and Michelson contrast for achromatic calculations. At 20 m depth, T. delaisi iris radiance generated strong achromatic contrasts against substrate radiance, regardless of exposure, and despite substrate fluorescence. Given that downwelling light above 600 nm is negligible at this depth, we can attribute this effect to iris fluorescence. Contrasts were weaker in 5 m. Yet, the pooled radiance caused by red reflection and fluorescence still exceeded substrate radiance for all substrates under shaded conditions and all but Jania rubens and Padina pavonia under exposed conditions. Due to the negative effects of anesthesia on iris fluorescence, these estimates are conservative. We conclude that the requirements to create visual brightness contrasts are fulfilled for a wide range of conditions in the natural environment of T. delaisi .

Three-dimensional quick response code based on inkjet printing of upconversion fluorescent nanoparticles for drug anti-counterfeiting

NASA Astrophysics Data System (ADS)

You, Minli; Lin, Min; Wang, Shurui; Wang, Xuemin; Zhang, Ge; Hong, Yuan; Dong, Yuqing; Jin, Guorui; Xu, Feng

2016-05-01

Medicine counterfeiting is a serious issue worldwide, involving potentially devastating health repercussions. Advanced anti-counterfeit technology for drugs has therefore aroused intensive interest. However, existing anti-counterfeit technologies are associated with drawbacks such as the high cost, complex fabrication process, sophisticated operation and incapability in authenticating drug ingredients. In this contribution, we developed a smart phone recognition based upconversion fluorescent three-dimensional (3D) quick response (QR) code for tracking and anti-counterfeiting of drugs. We firstly formulated three colored inks incorporating upconversion nanoparticles with RGB (i.e., red, green and blue) emission colors. Using a modified inkjet printer, we printed a series of colors by precisely regulating the overlap of these three inks. Meanwhile, we developed a multilayer printing and splitting technology, which significantly increases the information storage capacity per unit area. As an example, we directly printed the upconversion fluorescent 3D QR code on the surface of drug capsules. The 3D QR code consisted of three different color layers with each layer encoded by information of different aspects of the drug. A smart phone APP was designed to decode the multicolor 3D QR code, providing the authenticity and related information of drugs. The developed technology possesses merits in terms of low cost, ease of operation, high throughput and high information capacity, thus holds great potential for drug anti-counterfeiting.Medicine counterfeiting is a serious issue worldwide, involving potentially devastating health repercussions. Advanced anti-counterfeit technology for drugs has therefore aroused intensive interest. However, existing anti-counterfeit technologies are associated with drawbacks such as the high cost, complex fabrication process, sophisticated operation and incapability in authenticating drug ingredients. In this contribution, we developed a smart phone recognition based upconversion fluorescent three-dimensional (3D) quick response (QR) code for tracking and anti-counterfeiting of drugs. We firstly formulated three colored inks incorporating upconversion nanoparticles with RGB (i.e., red, green and blue) emission colors. Using a modified inkjet printer, we printed a series of colors by precisely regulating the overlap of these three inks. Meanwhile, we developed a multilayer printing and splitting technology, which significantly increases the information storage capacity per unit area. As an example, we directly printed the upconversion fluorescent 3D QR code on the surface of drug capsules. The 3D QR code consisted of three different color layers with each layer encoded by information of different aspects of the drug. A smart phone APP was designed to decode the multicolor 3D QR code, providing the authenticity and related information of drugs. The developed technology possesses merits in terms of low cost, ease of operation, high throughput and high information capacity, thus holds great potential for drug anti-counterfeiting. Electronic supplementary information (ESI) available: Calculating details of UCNP content per 3D QR code and decoding process of the 3D QR code. See DOI: 10.1039/c6nr01353h

A photoswitchable orange-to-far-red fluorescent protein, PSmOrange.

PubMed

Subach, Oksana M; Patterson, George H; Ting, Li-Min; Wang, Yarong; Condeelis, John S; Verkhusha, Vladislav V

2011-07-31

We report a photoswitchable monomeric Orange (PSmOrange) protein that is initially orange (excitation, 548 nm; emission, 565 nm) but becomes far-red (excitation, 636 nm; emission, 662 nm) after irradiation with blue-green light. Compared to its parental orange proteins, PSmOrange has greater brightness, faster maturation, higher photoconversion contrast and better photostability. The red-shifted spectra of both forms of PSmOrange enable its simultaneous use with cyan-to-green photoswitchable proteins to study four intracellular populations. Photoconverted PSmOrange has, to our knowledge, the most far-red excitation peak of all GFP-like fluorescent proteins, provides diffraction-limited and super-resolution imaging in the far-red light range, is optimally excited with common red lasers, and can be photoconverted subcutaneously in a mouse. PSmOrange photoswitching occurs via a two-step photo-oxidation process, which causes cleavage of the polypeptide backbone. The far-red fluorescence of photoconverted PSmOrange results from a new chromophore containing N-acylimine with a co-planar carbon-oxygen double bond.

Red-emitting fluorescent probe for detecting hypochlorite acid in vitro and in vivo.

PubMed

Chen, Hong; Sun, Tao; Qiao, Xiao-Guang; Tang, Qian-Oian; Zhao, Shan-Chao; Zhou, Zhan

2018-06-12

Due to the importance of hypochlorous acid (HClO) in biological and industrial, development of fluorescent probes for HClO has been an active research area. Here, a new red-emitting ratiometric fluorescent probe (P) was synthesized and well defined characterization via NMR, HR-MS, and fluorescence spectrum, which serves as a selective and sensitive probe for ClO - group. The probe showed a ratiometric fluorescent response to hypochlorite at the emission intensities ratio (I 480 /I 612 ) increasing from 0.28 to 27.46. The emission intensities ratio (I 480 /I 612 ) was linearly enhanced (I 480 /I 612  = 0.064 X + 0.096) with the ClO - concentration range from 1 to 30 μM. The detection limitation for ClO - in aqueous solution is 0.47 μM. Moreover, this biocompatible red-emitting ratiometric fluorescent probe was utilized to the fluorescence imaging of ClO - in living cells and Zebrafish. Copyright © 2018. Published by Elsevier B.V.

Delocalization of frontier orbitals induced red emission for heptazine based thermally activated delayed fluorescence molecule: First-principles study

NASA Astrophysics Data System (ADS)

Kang, Yongxiang; Zhao, Liyun; Leng, Jiancai

2018-04-01

Design of red organic emitting molecules with characteristic of thermally activated delayed fluorescence (TADF) remains a great challenge. Here, electronic and optical properties of a series of multi-branched TADF molecules have been investigated based on the newly-proposed optimal Hartree-Fock percentage method. Results show that, though enlarging the delocalization of HOMO and LUMO, the emission wavelength is redshift. The designed red TADF molecule possesses smaller reorganization energy than these for reported molecules. This indicates the non-radiative energy consumption of excited state is small and effective luminescence can be expected. Thus, a promising red thermally activated delayed fluorescence molecule is proposed.

Recovery of Red Fluorescent Protein Chromophore Maturation Deficiency through Rational Design

PubMed Central

Moore, Matthew M.; Oteng-Pabi, Samuel K.; Pandelieva, Antonia T.; Mayo, Stephen L.; Chica, Roberto A.

2012-01-01

Red fluorescent proteins (RFPs) derived from organisms in the class Anthozoa have found widespread application as imaging tools in biological research. For most imaging experiments, RFPs that mature quickly to the red chromophore and produce little or no green chromophore are most useful. In this study, we used rational design to convert a yellow fluorescent mPlum mutant to a red-emitting RFP without reverting any of the mutations causing the maturation deficiency and without altering the red chromophore’s covalent structure. We also created an optimized mPlum mutant (mPlum-E16P) that matures almost exclusively to the red chromophore. Analysis of the structure/function relationships in these proteins revealed two structural characteristics that are important for efficient red chromophore maturation in DsRed-derived RFPs. The first is the presence of a lysine residue at position 70 that is able to interact directly with the chromophore. The second is an absence of non-bonding interactions limiting the conformational flexibility at the peptide backbone that is oxidized during red chromophore formation. Satisfying or improving these structural features in other maturation-deficient RFPs may result in RFPs with faster and more complete maturation to the red chromophore. PMID:23285050

Spectral Diversity and Regulation of Coral Fluorescence in a Mesophotic Reef Habitat in the Red Sea.

PubMed

Eyal, Gal; Wiedenmann, Jörg; Grinblat, Mila; D'Angelo, Cecilia; Kramarsky-Winter, Esti; Treibitz, Tali; Ben-Zvi, Or; Shaked, Yonathan; Smith, Tyler B; Harii, Saki; Denis, Vianney; Noyes, Tim; Tamir, Raz; Loya, Yossi

2015-01-01

The phenomenon of coral fluorescence in mesophotic reefs, although well described for shallow waters, remains largely unstudied. We found that representatives of many scleractinian species are brightly fluorescent at depths of 50-60 m at the Interuniversity Institute for Marine Sciences (IUI) reef in Eilat, Israel. Some of these fluorescent species have distribution maxima at mesophotic depths (40-100 m). Several individuals from these depths displayed yellow or orange-red fluorescence, the latter being essentially absent in corals from the shallowest parts of this reef. We demonstrate experimentally that in some cases the production of fluorescent pigments is independent of the exposure to light; while in others, the fluorescence signature is altered or lost when the animals are kept in darkness. Furthermore, we show that green-to-red photoconversion of fluorescent pigments mediated by short-wavelength light can occur also at depths where ultraviolet wavelengths are absent from the underwater light field. Intraspecific colour polymorphisms regarding the colour of the tissue fluorescence, common among shallow water corals, were also observed for mesophotic species. Our results suggest that fluorescent pigments in mesophotic reefs fulfil a distinct biological function and offer promising application potential for coral-reef monitoring and biomedical imaging.

Complementary standoff chemical imaging to map and identify artist materials in an early Italian Renaissance panel painting.

PubMed

Dooley, Kathryn A; Conover, Damon M; Glinsman, Lisha Deming; Delaney, John K

2014-12-08

Two imaging modalities based on molecular and elemental spectroscopy were used to characterize a painting by Cosimo Tura. Visible-to-near-infrared (400-1680 nm) reflectance imaging spectroscopy (RIS) and X-ray fluorescence (XRF) imaging spectroscopy were employed to identify pigments and determine their spatial distribution with higher confidence than from either technique alone. For example, Mary's red robe was modeled through the distribution of an insect-derived red lake (RIS map) and lead white (XRF lead map), rather than a layer of red lake on vermilion. The RIS image cube was also used to isolate the preparatory design by mapping the reflectance spectra associated with it. In conjunction with results from an earlier RIS study (1650-2500 nm) to map and identify the binding media, a more thorough understanding was gained of the materials and techniques used in the painting. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Room temperature spectrally resolved single-molecule spectroscopy reveals new spectral forms and photophysical versatility of aequorea green fluorescent protein variants.

PubMed

Blum, Christian; Meixner, Alfred J; Subramaniam, Vinod

2004-12-01

It is known from ensemble spectroscopy at cryogenic temperatures that variants of the Aequorea green fluorescent protein (GFP) occur in interconvertible spectroscopically distinct forms which are obscured in ensemble room temperature spectroscopy. By analyzing the fluorescence of the GFP variants EYFP and EGFP by spectrally resolved single-molecule spectroscopy we were able to observe spectroscopically different forms of the proteins and to dynamically monitor transitions between these forms at room temperature. In addition to the predominant EYFP B-form we have observed the blue-shifted I-form thus far only seen at cryogenic temperatures and have followed transitions between these forms. Further we have identified for EYFP and for EGFP three more, so far unknown, forms with red-shifted fluorescence. Transitions between the predominant forms and the red-shifted forms show a dark time which indicates the existence of a nonfluorescent intermediate. The spectral position of the newly-identified red-shifted forms and their formation via a nonfluorescent intermediate hint that these states may account for the possible photoactivation observed in bulk experiments. The comparison of the single-protein spectra of the red-shifted EYFP and EGFP forms with single-molecule fluorescence spectra of DsRed suggest that these new forms possibly originate from an extended chromophoric pi-system analogous to the DsRed chromophore.

Red-light excitation of protoporphyrin IX fluorescence for subsurface tumor detection.

PubMed

Roberts, David W; Olson, Jonathan D; Evans, Linton T; Kolste, Kolbein K; Kanick, Stephen C; Fan, Xiaoyao; Bravo, Jaime J; Wilson, Brian C; Leblond, Frederic; Marois, Mikael; Paulsen, Keith D

2018-06-01

OBJECTIVE The objective of this study was to detect 5-aminolevulinic acid (ALA)-induced tumor fluorescence from glioma below the surface of the surgical field by using red-light illumination. METHODS To overcome the shallow tissue penetration of blue light, which maximally excites the ALA-induced fluorophore protoporphyrin IX (PpIX) but is also strongly absorbed by hemoglobin and oxyhemoglobin, a system was developed to illuminate the surgical field with red light (620-640 nm) matching a secondary, smaller absorption peak of PpIX and detecting the fluorescence emission through a 650-nm longpass filter. This wide-field spectroscopic imaging system was used in conjunction with conventional blue-light fluorescence for comparison in 29 patients undergoing craniotomy for resection of high-grade glioma, low-grade glioma, meningioma, or metastasis. RESULTS Although, as expected, red-light excitation is less sensitive to PpIX in exposed tumor, it did reveal tumor at a depth up to 5 mm below the resection bed in 22 of 24 patients who also exhibited PpIX fluorescence under blue-light excitation during the course of surgery. CONCLUSIONS Red-light excitation of tumor-associated PpIX fluorescence below the surface of the surgical field can be achieved intraoperatively and enables detection of subsurface tumor that is not visualized under conventional blue-light excitation. Clinical trial registration no.: NCT02191488 (clinicaltrials.gov).

Connoted hazard and perceived importance of fluorescent, neon, and standard safety colors.

PubMed

Zielinska, O A; Mayhorn, C B; Wogalter, M S

2017-11-01

The perceived hazard and rated importance of standard safety, fluorescent, and neon colors are investigated. Colors are used in warnings to enhance hazard communication. Red has consistently been rated as the highest in perceived hazard. Orange, yellow, and black are the next highest in connoted hazard; however, there is discrepancy in their ordering. Safety standards, such as ANSI Z535.1, also list colors to convey important information, but little research has examined the perceived importance of colors. In addition to standard safety colors, fluorescent colors are more commonly used in warnings. Understanding hazard and importance perceptions of standard safety and fluorescent colors is necessary to create effective warnings. Ninety participants rated and ranked a total of 33 colors on both perceived hazard and perceived importance. Rated highest were the safety red colors from the American National Standard Institute (ANSI), International Organization for Standardization (ISO), and Federal Highway Administration (FHWA) together with three fluorescent colors (orange, yellow, and yellow-green) from 3 M on both dimensions. Rankings were similar to ratings except that fluorescent orange was the highest on perceived hazard, while fluorescent orange and safety red from the ANSI were ranked as the highest in perceived importance. Fluorescent colors convey hazard and importance levels as high as the standard safety red colors. Implications for conveying hazard and importance in warnings through color are discussed. Copyright © 2017 Elsevier Ltd. All rights reserved.

Protein labeling with red squarylium dyes for analysis by capillary electrophoresis with laser-induced fluorescence detection.

PubMed

Yan, Weiying; Sloat, Amy L; Yagi, Shigeyuki; Nakazumi, Hiroyuki; Colyer, Christa L

2006-04-01

Two new red luminescent asymmetric squarylium dyes (designated "Red-1c and Red-3") have been shown to exhibit absorbance shifts to longer wavelengths upon the addition of protein, along with a concomitant increase in fluorescence emission. Specifically, the absorbance maxima for Red-1c and Red-3 dyes are 607 and 622 nm, respectively, in the absence of HSA, and 642 and 640 nm in the presence of HSA, making the excitation of their protein complexes feasible with inexpensive and robust diode lasers. Fluorescence emission maxima, in the presence of HSA, are 656 and 644 nm for Red-1c and Red-3, respectively. Because of the inherently low fluorescence of the dyes in their free state, Red-1c and Red-3 were used as on-column labels (that is, with the dye incorporated into the separation buffer), thus eliminating the need for sample derivatization prior to injection and separation. A comparison of precolumn and on-column labeling of proteins with these squarylium dyes revealed higher efficiencies and greater sensitivities for on-column labeling, which, when conducted with a basic, high-salt content buffer, permitted baseline resolution of a mixture of five model proteins. LOD for model proteins, such as transferrin, alpha-lactalbumin, BSA, and beta-lactoglobulin A and B, labeled with these dyes and analyzed by CE with LIF detection (CE-LIF) were found to be dependent upon dye concentration and solution pH, and are as low as 5 nM for BSA. Satisfactory linear relationships between peak height (or peak area) and protein concentration were obtained by CE-LIF for this on-column labeling method with Red-3 and Red-1c.

Conformational fluctuations in a green fluorescent protein-like Akane family protein: a high-pressure fluorescence study at 0.1-700 MPa

NASA Astrophysics Data System (ADS)

Maeno, Akihiro; Kato, Yuko; Jimbo, Mitsuru; Amada, Kei; Mita, Hajime; Akasaka, Kazuyuki

2017-04-01

We have investigated conformational fluctuations in a new green fluorescent protein(GFP)-like protein rb-Akane found in a red-brown-colored octocoral, Scleronephthya gracillima (Kuekenthal)), with high pressure fluorescence spectroscopy at 0.1-700 MPa. Besides the green fluorescence at 510 nm, two red fluorescence peaks are observed at 590 and 629 nm, the relative intensity of which varies reversibly with pressure. The phenomenon is interpreted as representing the cis-trans isomerization of the chromophore accompanied by the conformational transition between two sub-states of the red fluorescence form of rb-Akane. The two sub-states are separated only marginally in free energy (ΔG0 = 1.9 ± 0.4 kJ mol-1), but significantly in partial molar volume (ΔV0 = -19.8 ± 1.4 ml mol-1) at 0.1 MPa (pH 7.5, 25°C). Above 500 MPa, the fluorescence at λmax 629 nm undergoes another reversible change with pressure, showing the onset of unfolding.

Organimetallic Fluorescent Complex Polymers For Light Emitting Applications

DOEpatents

Shi, Song Q.; So, Franky

1997-10-28

A fluorescent complex polymer with fluorescent organometallic complexes connected by organic chain spacers is utilized in the fabrication of light emitting devices on a substantially transparent planar substrate by depositing a first conductive layer having p-type conductivity on the planar surface of the substrate, depositing a layer of a hole transporting and electron blocking material on the first conductive layer, depositing a layer of the fluorescent complex polymer on the layer of hole transporting and electron blocking material as an electron transporting emissive layer and depositing a second conductive layer having n-type conductivity on the layer of fluorescent complex polymer.

Current density reversibly alters metabolic spatial structure of exoelectrogenic anode biofilms

NASA Astrophysics Data System (ADS)

Sun, Dan; Cheng, Shaoan; Zhang, Fang; Logan, Bruce E.

2017-07-01

Understanding how current densities affect electrogenic biofilm activity is important for wastewater treatment as current densities can substantially decrease at COD concentrations greater than those suitable for discharge to the environment. We examined the biofilm's response, in terms of viability and enzymatic activity, to different current densities using microbial electrolysis cells with a lower (0.7 V) or higher (0.9 V) added voltage to alter current production. Viability was assessed using florescent dyes, with dead cells identified on the basis of dye penetration due to a compromised cell outer-membrane (red), and live cells (intact membrane) fluorescing green. Biofilms operated with 0.7 V produced 2.4 ± 0.2 A m-2, and had an inactive layer near the electrode and a viable layer at the biofilm-solution interface. The lack of cell activity near the electrode surface was confirmed by using an additional dye that fluoresces only with enzymatic activity. Adding 0.9 V increased the current by 61%, and resulted in a single, more homogeneous and active biofilm layer. Switching biofilms between these two voltages produced outcomes associated with the new current rather than the previous biofilm conditions. These findings suggest that maintaining higher current densities will be needed to ensure long-term viability electrogenic biofilms.

Strategy for synthesizing quantum dot-layered double hydroxide nanocomposites and their enhanced photoluminescence and photostability.

PubMed

Cho, Seungho; Jung, Sungwook; Jeong, Sanghwa; Bang, Jiwon; Park, Joonhyuck; Park, Youngrong; Kim, Sungjee

2013-01-08

Layered double hydroxide-quantum dot (LDH-QD) composites are synthesized via a room temperature LDH formation reaction in the presence of QDs. InP/ZnS (core/shell) QD, a heavy metal free QD, is used as a model constituent. Interactions between QDs (with negative zeta potentials), decorated with dihydrolipoic acids, and inherently positively charged metal hydroxide layers of LDH during the LDH formations are induced to form the LDH-QD composites. The formation of the LDH-QD composites affords significantly enhanced photoluminescence quantum yields and thermal- and photostabilities compared to their QD counterparts. In addition, the fluorescence from the solid LDH-QD composite preserved the initial optical properties of the QD colloid solution without noticeable deteriorations such as red-shift or deep trap emission. Based on their advantageous optical properties, we also demonstrate the pseudo white light emitting diode, down-converted by the LDH-QD composites.

Crystal structure of the fluorescent protein from Dendronephthya sp. in both green and photoconverted red forms

DOE Office of Scientific and Technical Information (OSTI.GOV)

Pletneva, Nadya V.; Pletnev, Sergei; Pakhomov, Alexey A.

The fluorescent protein fromDendronephthyasp. (DendFP) is a member of the Kaede-like group of photoconvertible fluorescent proteins with a His62-Tyr63-Gly64 chromophore-forming sequence. Upon irradiation with UV and blue light, the fluorescence of DendFP irreversibly changes from green (506 nm) to red (578 nm). The photoconversion is accompanied by cleavage of the peptide backbone at the C α—N bond of His62 and the formation of a terminal carboxamide group at the preceding Leu61. The resulting double C α=C βbond in His62 extends the conjugation of the chromophore π system to include imidazole, providing the red fluorescence. Here, the three-dimensional structures of nativemore » green and photoconverted red forms of DendFP determined at 1.81 and 2.14 Å resolution, respectively, are reported. This is the first structure of photoconverted red DendFP to be reported to date. The structure-based mutagenesis of DendFP revealed an important role of positions 142 and 193: replacement of the original Ser142 and His193 caused a moderate red shift in the fluorescence and a considerable increase in the photoconversion rate. It was demonstrated that hydrogen bonding of the chromophore to the Gln116 and Ser105 cluster is crucial for variation of the photoconversion rate. The single replacement Gln116Asn disrupts the hydrogen bonding of Gln116 to the chromophore, resulting in a 30-fold decrease in the photoconversion rate, which was partially restored by a further Ser105Asn replacement.« less

Red fluorescent proteins (RFPs) and RFP-based biosensors for neuronal imaging applications

PubMed Central

Shen, Yi; Lai, Tiffany; Campbell, Robert E.

2015-01-01

Abstract. The inherent advantages of red-shifted fluorescent proteins and fluorescent protein-based biosensors for the study of signaling processes in neurons and other tissues have motivated the development of a plethora of new tools. Relative to green fluorescent proteins (GFPs) and other blue-shifted alternatives, red fluorescent proteins (RFPs) provide the inherent advantages of lower phototoxicity, lower autofluorescence, and deeper tissue penetration associated with longer wavelength excitation light. All other factors being the same, the multiple benefits of using RFPs make these tools seemingly ideal candidates for use in neurons and, ultimately, the brain. However, for many applications, the practical utility of RFPs still falls short of the preferred GFPs. We present an overview of RFPs and RFP-based biosensors, with an emphasis on their reported applications in neuroscience. PMID:26158012

Development of a neutral embedding resin for optical imaging of fluorescently labeled biological tissue

NASA Astrophysics Data System (ADS)

Zhou, Hongfu; Gang, Yadong; Chen, Shenghua; Wang, Yu; Xiong, Yumiao; Li, Longhui; Yin, Fangfang; Liu, Yue; Liu, Xiuli; Zeng, Shaoqun

2017-10-01

Plastic embedding is widely applied in light microscopy analyses. Previous studies have shown that embedding agents and related techniques can greatly affect the quality of biological tissue embedding and fluorescent imaging. Specifically, it is difficult to preserve endogenous fluorescence using currently available acidic commercial embedding resins and related embedding techniques directly. Here, we developed a neutral embedding resin that improved the green fluorescent protein (GFP), yellow fluorescent protein (YFP), and DsRed fluorescent intensity without adjusting the pH value of monomers or reactivating fluorescence in lye. The embedding resin had a high degree of polymerization, and its fluorescence preservation ratios for GFP, YFP, and DsRed were 126.5%, 155.8%, and 218.4%, respectively.

Rapid and Sensitive Detection of Cardiac Troponin I for Point-of-Care Tests Based on Red Fluorescent Microspheres.

PubMed

Cai, Yanxue; Kang, Keren; Li, Qianru; Wang, Yu; He, Xiaowei

2018-05-07

A reliable lateral flow immunoassay (LFIA) based on a facile one-step synthesis of single microspheres in combining with immunochromatography technique was developed to establish a new point-of-care test (POCT) for the rapid and early detection of cardiac troponin I (cTnI), a kind of cardiac specific biomarker for acute myocardial infarction (AMI). The double layered microspheres with clear core-shell structures were produced using soap-free emulsion polymerization method with inexpensive compounds (styrene and acrylic acid). The synthetic process was simple, rapid and easy to control due to one-step synthesis without any complicated procedures. The microspheres are nanostructure with high surface area, which have numerous carboxyl groups on the out layer, resulting in high-efficiency coupling between the carrier and antibody via amide bond. Meanwhile, the red fluorescent dye, Nile-red (NR), was wrapped inside the microspheres to improve its stability, as well to reduce the background noise, because of its higher emission wavelength than interference from real plasma samples. The core-shell structures provided different functional areas to separate antibody and dyes, so the immunoassay has highly sensitive, wide working curves in the range of 0⁻40 ng/mL, low limits of detection (LOD) at 0.016 ng/mL, and limits of quantification (LOQ) at 0.087 ng/mL with coefficient of variations (CV) of 10%. This strategy suggested an outstanding platform for LFIA, with good reproducibility and stability to straightforwardly analyze the plasma samples without washing steps, thereby reducing the operating procedures for non-professionals and promoting detection efficiency. The whole detection process can be completed in less than 15 min. This novel immunoassay offers a reliable and favorable analytical result by detecting the real samples, indicating that it holds great potential as a new alternative for biomolecule detection in complex samples, for the early detection of cardiac specific biomarkers.

Enhanced fluorescence of epicocconone in surfactant assemblies as a consequence of depth-dependent microviscosity.

PubMed

Panda, Debashis; Khatua, Saumyakanti; Datta, Anindya

2007-02-22

The extents of fluorescence enhancement of epicocconone are found to be different in the micelles of the surfactants sodium dodecyl sulfate (SDS) and Triton X100 (TX 100). A decrease in fluorescence, observed in the cationic cetyltrimethylammonium bromide (CTAB) micelles, is rationalized by the formation of anions of the fluorophore at the Stern layer. To understand the difference in the effects of SDS and TX 100, the nature of the excited-state process in the fluorophore has been investigated by fluorescence spectroscopy, supported by complementary quantum chemical calculations. The excited-state dynamics of epicocconone is found to depend on polarity and viscosity of the medium, with a more pronounced dependence on viscosity. An inspection of the molecular orbitals involved in the electronic absorption of the molecule reveals the possibility of photoisomerization, which conforms to the observed solvent dependence of the fluorescence spectral properties. An apparent mismatch between trends observed in steady-state spectra and those in temporal decays indicates a significant contribution of an ultrafast component, which cannot be detected in the time resolution of our instrument. The viscosity dependence of the fluorescence quantum yields provides an explanation for the difference in the extents of fluorescence enhancement in the two micelles, in the light of location of the fluorophore at different depths of the micelle. The enhancement of fluorescence, with an unchanged fluorescence maximum, opens up the possibility that the fluorophore could be a useful dual emitting marker for fluorescence microscopy of heterogeneous systems, as the fluorescence of protein-bound epicocconone has been previously reported to be significantly red-shifted.

Enhanced emission of Nile Red on plasmonic platforms

NASA Astrophysics Data System (ADS)

Synak, Anna; Bojarski, Piotr; Grobelna, Beata; Gryczyński, Ignacy; Fudala, Rafał; Mońka, Michal

2018-04-01

Strongly enhanced fluorescence of Nile Red deposited in the vicinity of silver nanoparticles and gold semitransparent mirror was observed. The properties of three different plasmonic platforms based on TiO2, TiO2-GLYMO (1:1) and SiO2 matrices were studied with spectroscopic and microscopic techniques. Significant differences of Nile Red spectroscopic properties in both matrices were observed. In particular, the sensitivity of Nile Red fluorescence enhancement and its peak location to the polarity of local surrounding was found.

The fluorescence theatre: a cost-effective device using theatre gels for fluorescent protein and dye screening.

PubMed

Heil, John R; Nordeste, Ricardo F; Charles, Trevor C

2011-04-01

Here we report a simple cost-effective device for screening colonies on plates for expression of the monomeric red fluorescent protein mRFP1 and the fluorescent dye Nile red. This device can be built from any simple light source, in our case a Quebec Colony Counter, and cost-effective theatre gels. The device can be assembled in as little as 20 min, and it produces excellent results when screening a large number of colonies.

a Model to Simulate the Radiative Transfer of Fluorescence in a Leaf

NASA Astrophysics Data System (ADS)

Zhao, F.; Ni, Q.

2018-04-01

Light is reflected, transmitted and absorbed by green leaves. Chlorophyll fluorescence (ChlF) is the signal emitted by chlorophyll molecules in the leaf after the absorption of light. ChlF can be used as a direct probe of the functional status of photosynthetic machinery because of its close relationship with photosynthesis. The scattering, absorbing, and emitting properties of leaves are spectrally dependent, which can be simulated by modeling leaf-level fluorescence. In this paper, we proposed a Monte-Carlo (MC) model to simulate the radiative transfer of photons in the leaf. Results show that typical leaf fluorescence spectra can be properly simulated, with two peaks centered at around 685 nm in the red and 740 nm in the far-red regions. By analysing the sensitivity of the input parameters, we found the MC model can well simulate their influence on the emitted fluorescence. Meanwhile we compared results simulated by MC model with those by the Fluspect model. Generally they agree well in the far-red region but deviate in the red region.

An ensemble and single-molecule fluorescence microscopy investigation of phase-separated monolayer films stained with Nile Red.

PubMed

Lu, Yin; Porterfield, Robyn; Thunder, Terri; Paige, Matthew F

2011-01-01

Phase-separated Langmuir-Blodgett monolayer films prepared from mixtures of arachidic acid (C19H39COOH) and perfluorotetradecanoic acid (C13F27COOH) were stained via spin-casting with the polarity sensitive phenoxazine dye Nile Red, and characterized using a combination of ensemble and single-molecule fluorescence microscopy measurements. Ensemble fluorescence microscopy and spectromicroscopy showed that Nile Red preferentially associated with the hydrogenated domains of the phase-separated films, and was strongly fluorescent in these areas of the film. These measurements, in conjunction with single-molecule fluorescence imaging experiments, also indicated that a small sub-population of dye molecules localizes on the perfluorinated regions of the sample, but that this sub-population is spectroscopically indistinguishable from that associated with the hydrogenated domains. The relative importance of selective dye adsorption and local polarity sensitivity of Nile Red for staining applications in phase-separated LB films as well as in cellular environments is discussed in context of the experimental results. Copyright © 2010 Elsevier B.V. All rights reserved.

A Bright and Fast Red Fluorescent Protein Voltage Indicator That Reports Neuronal Activity in Organotypic Brain Slices

PubMed Central

Abdelfattah, Ahmed S.; Farhi, Samouil L.; Zhao, Yongxin; Brinks, Daan; Zou, Peng; Ruangkittisakul, Araya; Platisa, Jelena; Pieribone, Vincent A.; Ballanyi, Klaus; Cohen, Adam E.

2016-01-01

Optical imaging of voltage indicators based on green fluorescent proteins (FPs) or archaerhodopsin has emerged as a powerful approach for detecting the activity of many individual neurons with high spatial and temporal resolution. Relative to green FP-based voltage indicators, a bright red-shifted FP-based voltage indicator has the intrinsic advantages of lower phototoxicity, lower autofluorescent background, and compatibility with blue-light-excitable channelrhodopsins. Here, we report a bright red fluorescent voltage indicator (fluorescent indicator for voltage imaging red; FlicR1) with properties that are comparable to the best available green indicators. To develop FlicR1, we used directed protein evolution and rational engineering to screen libraries of thousands of variants. FlicR1 faithfully reports single action potentials (∼3% ΔF/F) and tracks electrically driven voltage oscillations at 100 Hz in dissociated Sprague Dawley rat hippocampal neurons in single trial recordings. Furthermore, FlicR1 can be easily imaged with wide-field fluorescence microscopy. We demonstrate that FlicR1 can be used in conjunction with a blue-shifted channelrhodopsin for all-optical electrophysiology, although blue light photoactivation of the FlicR1 chromophore presents a challenge for applications that require spatially overlapping yellow and blue excitation. SIGNIFICANCE STATEMENT Fluorescent-protein-based voltage indicators enable imaging of the electrical activity of many genetically targeted neurons with high spatial and temporal resolution. Here, we describe the engineering of a bright red fluorescent protein-based voltage indicator designated as FlicR1 (fluorescent indicator for voltage imaging red). FlicR1 has sufficient speed and sensitivity to report single action potentials and voltage fluctuations at frequencies up to 100 Hz in single-trial recordings with wide-field microscopy. Because it is excitable with yellow light, FlicR1 can be used in conjunction with blue-light-activated optogenetic actuators. However, spatially distinct patterns of optogenetic activation and voltage imaging are required to avoid fluorescence artifacts due to photoactivation of the FlicR1 chromophore. PMID:26911693

Green and Red Fluorescent Dyes for Translational Applications in Imaging and Sensing Analytes: A Dual‐Color Flag

PubMed Central

Oliveira, Elisabete; Bértolo, Emilia; Núñez, Cristina; Pilla, Viviane; Santos, Hugo M.; Fernández‐Lodeiro, Javier; Fernández‐Lodeiro, Adrian; Djafari, Jamila; Capelo, José Luis

2017-01-01

Abstract Red and green are two of the most‐preferred colors from the entire chromatic spectrum, and red and green dyes are widely used in biochemistry, immunohistochemistry, immune‐staining, and nanochemistry applications. Selective dyes with green and red excitable chromophores can be used in biological environments, such as tissues and cells, and can be irradiated with visible light without cell damage. This critical review, covering a period of five years, provides an overview of the most‐relevant results on the use of red and green fluorescent dyes in the fields of bio‐, chemo‐ and nanoscience. The review focuses on fluorescent dyes containing chromophores such as fluorescein, rhodamine, cyanine, boron–dipyrromethene (BODIPY), 7‐nitobenz‐2‐oxa‐1,3‐diazole‐4‐yl, naphthalimide, acridine orange, perylene diimides, coumarins, rosamine, Nile red, naphthalene diimide, distyrylpyridinium, benzophosphole P‐oxide, benzoresorufins, and tetrapyrrolic macrocycles. Metal complexes and nanomaterials with these dyes are also discussed. PMID:29318095

A rapid-screening approach to detect and quantify microplastics based on fluorescent tagging with Nile Red

NASA Astrophysics Data System (ADS)

Maes, Thomas; Jessop, Rebecca; Wellner, Nikolaus; Haupt, Karsten; Mayes, Andrew G.

2017-03-01

A new approach is presented for analysis of microplastics in environmental samples, based on selective fluorescent staining using Nile Red (NR), followed by density-based extraction and filtration. The dye adsorbs onto plastic surfaces and renders them fluorescent when irradiated with blue light. Fluorescence emission is detected using simple photography through an orange filter. Image-analysis allows fluorescent particles to be identified and counted. Magnified images can be recorded and tiled to cover the whole filter area, allowing particles down to a few micrometres to be detected. The solvatochromic nature of Nile Red also offers the possibility of plastic categorisation based on surface polarity characteristics of identified particles. This article details the development of this staining method and its initial cross-validation by comparison with infrared (IR) microscopy. Microplastics of different sizes could be detected and counted in marine sediment samples. The fluorescence staining identified the same particles as those found by scanning a filter area with IR-microscopy.

Far-Red Fluorescent Lipid-Polymer Probes for an Efficient Labeling of Enveloped Viruses.

PubMed

Lacour, William; Adjili, Salim; Blaising, Julie; Favier, Arnaud; Monier, Karine; Mezhoud, Sarra; Ladavière, Catherine; Place, Christophe; Pécheur, Eve-Isabelle; Charreyre, Marie-Thérèse

2016-08-01

Far-red emitting fluorescent lipid probes are desirable to label enveloped viruses, for their efficient tracking by optical microscopy inside autofluorescent cells. Most used probes are rapidly released from membranes, leading to fluorescence signal decay and loss of contrast. Here, water-soluble lipid-polymer probes are synthesized harboring hydrophilic or hydrophobic far-red emitting dyes, and exhibiting enhanced brightness. They efficiently label Hepatitis C Virus pseudotyped particles (HCVpp), more stably and reproducibly than commercial probes, and a strong fluorescence signal is observed with a high contrast. Labeling with such probes do not alter virion morphology, integrity, nor infectivity. Finally, it is shown by fluorescence microscopy that these probes enable efficient tracking of labeled HCVpp inside hepatocarcinoma cells used as model hepatocytes, in spite of their autofluorescence up to 700 nm. These novel fluorescent lipid-polymer probes should therefore enable a better characterization of early stages of infection of autofluorescent cells by enveloped viruses. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Red Fluorescent Carbon Nanoparticle-Based Cell Imaging Probe.

PubMed

Ali, Haydar; Bhunia, Susanta Kumar; Dalal, Chumki; Jana, Nikhil R

2016-04-13

Fluorescent carbon nanoparticle-based probes with tunable visible emission are biocompatible, environment friendly and most suitable for various biomedical applications. However, synthesis of red fluorescent carbon nanoparticles and their transformation into functional nanoparticles are very challenging. Here we report red fluorescent carbon nanoparticle-based nanobioconjugates of <25 nm hydrodynamic size and their application as fluorescent cell labels. Hydrophobic carbon nanoparticles are synthesized via high temperature colloid-chemical approach and transformed into water-soluble functional nanoparticles via coating with amphiphilic polymer followed by covalent linking with desired biomolecules. Following this approach, carbon nanoparticles are functionalized with polyethylene glycol, primary amine, glucose, arginine, histidine, biotin and folic acid. These functional nanoparticles can be excited with blue/green light (i.e., 400-550 nm) to capture their emission spanning from 550 to 750 nm. Arginine and folic acid functionalized nanoparticles have been demonstrated as fluorescent cell labels where blue and green excitation has been used for imaging of labeled cells. The presented method can be extended for the development of carbon nanoparticle-based other bioimaging probes.

A rapid-screening approach to detect and quantify microplastics based on fluorescent tagging with Nile Red

PubMed Central

Maes, Thomas; Jessop, Rebecca; Wellner, Nikolaus; Haupt, Karsten; Mayes, Andrew G.

2017-01-01

A new approach is presented for analysis of microplastics in environmental samples, based on selective fluorescent staining using Nile Red (NR), followed by density-based extraction and filtration. The dye adsorbs onto plastic surfaces and renders them fluorescent when irradiated with blue light. Fluorescence emission is detected using simple photography through an orange filter. Image-analysis allows fluorescent particles to be identified and counted. Magnified images can be recorded and tiled to cover the whole filter area, allowing particles down to a few micrometres to be detected. The solvatochromic nature of Nile Red also offers the possibility of plastic categorisation based on surface polarity characteristics of identified particles. This article details the development of this staining method and its initial cross-validation by comparison with infrared (IR) microscopy. Microplastics of different sizes could be detected and counted in marine sediment samples. The fluorescence staining identified the same particles as those found by scanning a filter area with IR-microscopy. PMID:28300146

Spectral Diversity and Regulation of Coral Fluorescence in a Mesophotic Reef Habitat in the Red Sea

PubMed Central

Eyal, Gal; Wiedenmann, Jörg; Grinblat, Mila; D’Angelo, Cecilia; Kramarsky-Winter, Esti; Treibitz, Tali; Ben-Zvi, Or; Shaked, Yonathan; Smith, Tyler B.; Harii, Saki; Denis, Vianney; Noyes, Tim; Tamir, Raz; Loya, Yossi

2015-01-01

The phenomenon of coral fluorescence in mesophotic reefs, although well described for shallow waters, remains largely unstudied. We found that representatives of many scleractinian species are brightly fluorescent at depths of 50–60 m at the Interuniversity Institute for Marine Sciences (IUI) reef in Eilat, Israel. Some of these fluorescent species have distribution maxima at mesophotic depths (40–100 m). Several individuals from these depths displayed yellow or orange-red fluorescence, the latter being essentially absent in corals from the shallowest parts of this reef. We demonstrate experimentally that in some cases the production of fluorescent pigments is independent of the exposure to light; while in others, the fluorescence signature is altered or lost when the animals are kept in darkness. Furthermore, we show that green-to-red photoconversion of fluorescent pigments mediated by short-wavelength light can occur also at depths where ultraviolet wavelengths are absent from the underwater light field. Intraspecific colour polymorphisms regarding the colour of the tissue fluorescence, common among shallow water corals, were also observed for mesophotic species. Our results suggest that fluorescent pigments in mesophotic reefs fulfil a distinct biological function and offer promising application potential for coral-reef monitoring and biomedical imaging. PMID:26107282

A real-time single sperm tracking, laser trapping, and ratiometric fluorescent imaging system

NASA Astrophysics Data System (ADS)

Shi, Linda Z.; Botvinick, Elliot L.; Nascimento, Jaclyn; Chandsawangbhuwana, Charlie; Berns, Michael W.

2006-08-01

Sperm cells from a domestic dog were treated with oxacarbocyanine DiOC II(3), a ratiometrically-encoded membrane potential fluorescent probe in order to monitor the mitochondria stored in an individual sperm's midpiece. This dye normally emits a red fluorescence near 610 nm as well as a green fluorescence near 515 nm. The ratio of red to green fluorescence provides a substantially accurate and precise measurement of sperm midpiece membrane potential. A two-level computer system has been developed to quantify the motility and energetics of sperm using video rate tracking, automated laser trapping (done by the upper-level system) and fluorescent imaging (done by the lower-level system). The communication between these two systems is achieved by a networked gigabit TCP/IP cat5e crossover connection. This allows for the curvilinear velocity (VCL) and ratio of the red to green fluorescent images of individual sperm to be written to the hard drive at video rates. This two-level automatic system has increased experimental throughput over our previous single-level system (Mei et al., 2005) by an order of magnitude.

Intracellular zinc distribution in mitochondria, ER and the Golgi apparatus

PubMed Central

Lu, Qiping; Haragopal, Hariprakash; Slepchenko, Kira G; Stork, Christian; Li, Yang V

2016-01-01

Zinc (Zn2+) is required for numerous cellular functions. As such, the homeostasis and distribution of intracellular zinc can influence cellular metabolism and signaling. However, the exact distribution of free zinc within live cells remains elusive. Previously we showed the release of zinc from thapsigargin/IP3-sensitive endoplasmic reticulum (ER) storage in cortical neurons. In the present study, we investigated if other cellular organelles also contain free chelatable zinc and function as organelle storage for zinc. To identify free zinc within the organelles, live cells were co-stained with Zinpyr-1, a zinc fluorescent dye, and organelle-specific fluorescent dyes (MitoFluor Red 589: mitochondria; ER Tracker Red: endoplasmic reticulum; BODIPY TR ceramide: Golgi apparatus; Syto Red 64: nucleus). We examined organelles that represent potential storing sites for intracellular zinc. We showed that zinc fluorescence staining was co-localized with MitoFluor Red 589, ER Tracker Red, and BODIPY TR ceramide respectively, suggesting the presence of free zinc in mitochondria, endoplasmic reticulum, and the Golgi apparatus. On the other hand, cytosol and nucleus had nearly no detectable zinc fluorescence. It is known that nucleus contains high amount of zinc binding proteins that have high zinc binding affinity. The absence of zinc fluorescence suggests that there is little free zinc in these two regions. It also indicates that the zinc fluorescence detected in mitochondria, ER and Golgi apparatus represents free chelatable zinc. Taken together, our results support that these organelles are potential zinc storing organelles during cellular zinc homeostasis. PMID:27186321

Calcium Sensitive Fluorescent Dyes Fluo-4 and Fura Red under Pressure: Behaviour of Fluorescence and Buffer Properties under Hydrostatic Pressures up to 200 MPa.

PubMed

Schneidereit, D; Vass, H; Reischl, B; Allen, R J; Friedrich, O

2016-01-01

The fluorescent Ca2+ sensitive dyes Fura Red (ratiometric) and Fluo-4 (non-ratiometric) are widely utilized for the optical assessment of Ca2+ fluctuations in vitro as well as in situ. The fluorescent behavior of these dyes is strongly depends on temperature, pH, ionic strength and pressure. It is crucial to understand the response of these dyes to pressure when applying calcium imaging technologies in the field of high pressure bioscience. Therefore, we use an optically accessible pressure vessel to pressurize physiological Ca2+-buffered solutions at different fixed concentrations of free Ca2+ (1 nM to 25.6 μM) and a specified dye concentration (12 μM) to pressures of 200 MPa, and record dye fluorescence intensity. Our results show that Fluo-4 fluorescence intensity is reduced by 31% per 100 MPa, the intensity of Fura Red is reduced by 10% per 100 MPa. The mean reaction volume for the dissociation of calcium from the dye molecules [Formula: see text] is determined to -17.8 ml mol-1 for Fluo-4 and -21.3 ml mol-1 for Fura Red. Additionally, a model is presented that is used to correct for pressure-dependent changes in pH and binding affinity of Ca2+ to EGTA, as well as to determine the influence of these changes on dye fluorescence.

Three-dimensional quick response code based on inkjet printing of upconversion fluorescent nanoparticles for drug anti-counterfeiting.

PubMed

You, Minli; Lin, Min; Wang, Shurui; Wang, Xuemin; Zhang, Ge; Hong, Yuan; Dong, Yuqing; Jin, Guorui; Xu, Feng

2016-05-21

Medicine counterfeiting is a serious issue worldwide, involving potentially devastating health repercussions. Advanced anti-counterfeit technology for drugs has therefore aroused intensive interest. However, existing anti-counterfeit technologies are associated with drawbacks such as the high cost, complex fabrication process, sophisticated operation and incapability in authenticating drug ingredients. In this contribution, we developed a smart phone recognition based upconversion fluorescent three-dimensional (3D) quick response (QR) code for tracking and anti-counterfeiting of drugs. We firstly formulated three colored inks incorporating upconversion nanoparticles with RGB (i.e., red, green and blue) emission colors. Using a modified inkjet printer, we printed a series of colors by precisely regulating the overlap of these three inks. Meanwhile, we developed a multilayer printing and splitting technology, which significantly increases the information storage capacity per unit area. As an example, we directly printed the upconversion fluorescent 3D QR code on the surface of drug capsules. The 3D QR code consisted of three different color layers with each layer encoded by information of different aspects of the drug. A smart phone APP was designed to decode the multicolor 3D QR code, providing the authenticity and related information of drugs. The developed technology possesses merits in terms of low cost, ease of operation, high throughput and high information capacity, thus holds great potential for drug anti-counterfeiting.

Detection of TNT using a sensitive two-photon organic dendrimer for remote sensing

NASA Astrophysics Data System (ADS)

Narayanan, Aditya; Varnavski, Oleg; Mongin, Oliver; Majoral, Jean-Pierre; Blanchard-Desce, Mireille; Goodson, Theodore, III

2008-03-01

There is currently a need for superior stand-off detection schemes for protection against explosive weapons of mass destruction. Fluorescence detection at small distances from the target has proven to be attractive. A novel unexplored route in fluorescence chemical sensing that utilizes the exceptional spectroscopic capabilities of nonlinear optical methods is two-photon excited fluorescence. This approach utilizes infra-red light for excitation of remote sensors. Infra-red light suffers less scattering in porous materials which is beneficial for vapor sensing and has greater depth of penetration through the atmosphere, and there are fewer concerns regarding eye safety in remote detection schemes. We demonstrate this method using a novel dendritic system which possesses both excellent fluorescence sensitivity to the presence of TNT with infra-red pulses of light and high two-photon absorption (TPA) response. This illustrates the use of TPA for potential stand-off detection of energetic materials in the infra-red spectral regions in a highly two-photon responsive dendrimer.

Etching-dependent fluorescence quenching of Ag-dielectric-Au three-layered nanoshells: The effect of inner Ag nanosphere

NASA Astrophysics Data System (ADS)

Zhu, Jian; Xu, Zai-jie; Weng, Guo-jun; Zhao, Jing; Li, Jian-jun; Zhao, Jun-wu

2018-07-01

In this report, Ag-dielectric-Au three-layered nanoshells with controlled inner core size were synthesized. The fluorescence emission of the rhodamine 6G (R6G) could be quenched by the three-layered nanoshells distinctly. What's more, the fluorescence quenching efficiency could be further improved by tuning the etching of inner Ag nanosphere. The maximum fluorescence quenching efficiency is obtained when the separate layer just appears between the inner Ag core and the outer Au shell. Whereas the fluorescence quenching efficiency is weakened when no gaps take place around the inner Ag core or the separate layer is too thick and greater than 13 nm. The fluorescence quenching properties of the Ag-dielectric-Au three-layered nanoshells with different initial sizes of the Ag nanoparticles are also studied. The maximum fluorescence quenching efficiency is obtained when the three-layered nanoshells are synthesized based on the Ag nanoparticles with 60 nm, which is better than others two sizes (42 and 79 nm). Thus we believe that the size of initial Ag nanospheres also greatly affects the optimized fluorescence quenching efficiency. These results about fluorescence quenching properties of Ag-dielectric-Au three-layered nanoshells present a potential for design and fabrication of fluorescence nanosensors based on tuning the geometry of the inner core and the separate layer.

Red fluorescent proteins: advanced imaging applications and future design.

PubMed

Shcherbakova, Daria M; Subach, Oksana M; Verkhusha, Vladislav V

2012-10-22

In the past few years a large series of the advanced red-shifted fluorescent proteins (RFPs) has been developed. These enhanced RFPs provide new possibilities to study biological processes at the levels ranging from single molecules to whole organisms. Herein the relationship between the properties of the RFPs of different phenotypes and their applications to various imaging techniques are described. Existing and emerging imaging approaches are discussed for conventional RFPs, far-red FPs, RFPs with a large Stokes shift, fluorescent timers, irreversibly photoactivatable and reversibly photoswitchable RFPs. Advantages and limitations of specific RFPs for each technique are presented. Recent progress in understanding the chemical transformations of red chromophores allows the future RFP phenotypes and their respective novel imaging applications to be foreseen. Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Sensitive optical bio-sensing of p-type WSe2 hybridized with fluorescent dye attached DNA by doping and de-doping effects

NASA Astrophysics Data System (ADS)

Han, Kyu Hyun; Kim, Jun Young; Jo, Seong Gi; Seo, Changwon; Kim, Jeongyong; Joo, Jinsoo

2017-10-01

Layered transition metal dichalcogenides, such as MoS2, WSe2 and WS2, are exciting two-dimensional (2D) materials because they possess tunable optical and electrical properties that depend on the number of layers. In this study, the nanoscale photoluminescence (PL) characteristics of the p-type WSe2 monolayer, and WSe2 layers hybridized with the fluorescent dye Cy3 attached to probe-DNA (Cy3/p-DNA), have been investigated as a function of the concentration of Cy3/DNA by using high-resolution laser confocal microscopy. With increasing concentration of Cy3/p-DNA, the measured PL intensity decreases and its peak is red-shifted, suggesting that the WSe2 layer has been p-type doped with Cy3/p-DNA. Then, the PL intensity of the WSe2/Cy3/p-DNA hybrid system increases and the peak is blue-shifted through hybridization with relatively small amounts of target-DNA (t-DNA) (50-100 nM). This effect originates from charge and energy transfer from the Cy3/DNA to the WSe2. For t-DNA detection, our systems using p-type WSe2 have the merit in terms of the increase of PL intensity. The p-type WSe2 monolayers can be a promising nanoscale 2D material for sensitive optical bio-sensing based on the doping and de-doping responses to biomaterials.

Validation of a simple and fast method to quantify in vitro mineralization with fluorescent probes used in molecular imaging of bone

DOE Office of Scientific and Technical Information (OSTI.GOV)

Moester, Martiene J.C.; Schoeman, Monique A.E.; Oudshoorn, Ineke B.

2014-01-03

Highlights: •We validate a simple and fast method of quantification of in vitro mineralization. •Fluorescently labeled agents can detect calcium deposits in the mineralized matrix of cell cultures. •Fluorescent signals of the probes correlated with Alizarin Red S staining. -- Abstract: Alizarin Red S staining is the standard method to indicate and quantify matrix mineralization during differentiation of osteoblast cultures. KS483 cells are multipotent mouse mesenchymal progenitor cells that can differentiate into chondrocytes, adipocytes and osteoblasts and are a well-characterized model for the study of bone formation. Matrix mineralization is the last step of differentiation of bone cells and ismore » therefore a very important outcome measure in bone research. Fluorescently labelled calcium chelating agents, e.g. BoneTag and OsteoSense, are currently used for in vivo imaging of bone. The aim of the present study was to validate these probes for fast and simple detection and quantification of in vitro matrix mineralization by KS483 cells and thus enabling high-throughput screening experiments. KS483 cells were cultured under osteogenic conditions in the presence of compounds that either stimulate or inhibit osteoblast differentiation and thereby matrix mineralization. After 21 days of differentiation, fluorescence of stained cultures was quantified with a near-infrared imager and compared to Alizarin Red S quantification. Fluorescence of both probes closely correlated to Alizarin Red S staining in both inhibiting and stimulating conditions. In addition, both compounds displayed specificity for mineralized nodules. We therefore conclude that this method of quantification of bone mineralization using fluorescent compounds is a good alternative for the Alizarin Red S staining.« less

In situ X-ray fluorescence-based method to differentiate among red ochre pigments and yellow ochre pigments thermally transformed to red pigments of wall paintings from Pompeii.

PubMed

Marcaida, Iker; Maguregui, Maite; Fdez-Ortiz de Vallejuelo, Silvia; Morillas, Héctor; Prieto-Taboada, Nagore; Veneranda, Marco; Castro, Kepa; Madariaga, Juan Manuel

2017-06-01

Most of the magnificent wall paintings from the ancient city of Pompeii are decorated with red and yellow colors coming from the ochre pigments used. The thermal impact of the pyroclastic flow from the eruption of Vesuvius, in AD 79, promoted the transformation of some yellow painted areas to red. In this work, original red ochre, original yellow ochre, and transformed yellow ochre (nowadays showing a red color) of wall paintings from Pompeian houses (House of Marcus Lucretius and House of Gilded Cupids) were analyzed by means of a handheld energy-dispersive X-ray fluorescence spectrometer to develop a fast method that allows chemical differentiation of the original red ochre and the transformed yellow ochre. Principal component analysis of the multivariate obtained data showed that arsenic is the tracer element to distinguish between both red colored ochres. Moreover, Pompeian raw red and yellow ochre pigments recovered from the burial were analyzed in the laboratory with use of a benchtop energy-dispersive X-ray fluorescence spectrometer to confirm the elemental composition and the conclusions drawn from the in situ analysis according to the yellow ochre pigment transformation in real Pompeian wall paintings.

Fluorescence diffuse tomography of small animals with DsRed2 fluorescent protein

NASA Astrophysics Data System (ADS)

Turchin, I. V.; Plehanov, V. I.; Orlova, A. G.; Kamenskiy, V. A.; Kleshnin, M. S.; Shirmanova, M. V.; Shakhova, N. M.; Balalaeva, I. V.; Savitskiy, A. P.

2006-05-01

Fluorescent compounds are used as markers to diagnose oncological diseases, to study molecular processes typical for carcinogenesis, and to investigate metastasis formation and tumor regress under the influence of therapeutics. Different types of tomography, such as continuous wave (CW), frequency-domain (FD), and time-domain (TD) tomography, allow fluorescence imaging of tumors located deep in human or animal tissue. In this work, preliminary results of the frequency domain fluorescent diffuse tomography (FDT) method in application to DsRed2 protein as a fluorescent agent are presented. For the first step of our experiments, we utilized low-frequency amplitude modulation (1 kHz) of second harmonic of Nd: YAG (532 nm). The transilluminative configuration was used in the setup. The results of post mortem experiments with capsules containing DsRed2 inserted inside the esophagus of a 3-day-old hairless rat to simulate tumor are shown. An algorithm of processing fluorescent images based on calculating the zero of maximum curvature has been applied to detect fluorescent inclusion boundaries in the image. This work demonstrates the potential capability of the FDT method for imaging deep fluorescent tumors in human tissue or animal models of human cancer. Improvement of the setup can be accomplished by using high-frequency modulation (using a 110-MHz acoustooptical modulator).

Research on fluorescence detection method of Microcystis aeruginosa

NASA Astrophysics Data System (ADS)

Wang, Xiao-xiong

2017-07-01

The paper studied the viability determination of Microcystis aeruginosa by FDA and PI staining. The staining results were measured by fluorescence microscopy. The results indicated that viable and dead cells were stained as bright green and red fluorescent respectively by FDA and PI. Through PI-FDA dual color fluorescence staining, the color of green and red distinct obviously by fluorescence microscope. The staining rate has relation with the cell density. If the cell density of M. aeruginosa was 1.0×107-1.0×109 cell·mL-1, the staining rate would be 100.0% or 98.0% by PI and of FDA respectively.

Use of a night vision intensifier for direct visualization by eye of far-red and near-infrared fluorescence through an optical microscope.

PubMed

Siddiqi, M A; Kilduff, G M; Gearhart, J D

2003-11-01

We describe the design, construction and testing of a prototype device that allows the direct visualization by eye of far-red and near-infrared (NIR) fluorescence through an optical microscope. The device incorporates a gallium arsenide (GaAs) image intensifier, typically utilized in low-light or 'night vision' applications. The intensifier converts far-red and NIR light into electrons and then into green light, which is visible to the human eye. The prototype makes possible the direct, real-time viewing by eye of normally invisible far-red and NIR fluorescence from a wide variety of fluorophores, using the full field of view of the microscope to which it is applied. The high sensitivity of the image intensifier facilitates the viewing of a wide variety of photosensitive specimens, including live cells and embryos, at vastly reduced illumination levels in both fluorescence and bright-field microscopy. Modifications to the microscope are not required in order to use the prototype, which is fully compatible with all current fluorescence techniques. Refined versions of the prototype device will have broad research and clinical applications.

Red to far-red multispectral fluorescence image fusion for detection of fecal contamination on apples

USDA-ARS?s Scientific Manuscript database

This research developed a multispectral algorithm derived from hyperspectral line-scan fluorescence imaging under violet/blue LED excitation for detection of fecal contamination on Golden Delicious apples. Using a hyperspectral line-scan imaging system consisting of an EMCCD camera, spectrograph, an...

Calcium Sensitive Fluorescent Dyes Fluo-4 and Fura Red under Pressure: Behaviour of Fluorescence and Buffer Properties under Hydrostatic Pressures up to 200 MPa

PubMed Central

Vass, H.; Reischl, B.; Allen, R. J.; Friedrich, O.

2016-01-01

The fluorescent Ca2+ sensitive dyes Fura Red (ratiometric) and Fluo-4 (non-ratiometric) are widely utilized for the optical assessment of Ca2+ fluctuations in vitro as well as in situ. The fluorescent behavior of these dyes is strongly depends on temperature, pH, ionic strength and pressure. It is crucial to understand the response of these dyes to pressure when applying calcium imaging technologies in the field of high pressure bioscience. Therefore, we use an optically accessible pressure vessel to pressurize physiological Ca2+-buffered solutions at different fixed concentrations of free Ca2+ (1 nM to 25.6 μM) and a specified dye concentration (12 μM) to pressures of 200 MPa, and record dye fluorescence intensity. Our results show that Fluo-4 fluorescence intensity is reduced by 31% per 100 MPa, the intensity of Fura Red is reduced by 10% per 100 MPa. The mean reaction volume for the dissociation of calcium from the dye molecules Δdv¯ is determined to -17.8 ml mol-1 for Fluo-4 and -21.3 ml mol-1 for Fura Red. Additionally, a model is presented that is used to correct for pressure-dependent changes in pH and binding affinity of Ca2+ to EGTA, as well as to determine the influence of these changes on dye fluorescence. PMID:27764134

Engineering 'Golden' Fluorescence by Selective Pressure Incorporation of Non-canonical Amino Acids and Protein Analysis by Mass Spectrometry and Fluorescence.

PubMed

Baumann, Tobias; Schmitt, Franz-Josef; Pelzer, Almut; Spiering, Vivian Jeanette; Freiherr von Sass, Georg Johannes; Friedrich, Thomas; Budisa, Nediljko

2018-04-27

Fluorescent proteins are fundamental tools for the life sciences, in particular for fluorescence microscopy of living cells. While wild-type and engineered variants of the green fluorescent protein from Aequorea victoria (avGFP) as well as homologs from other species already cover large parts of the optical spectrum, a spectral gap remains in the near-infrared region, for which avGFP-based fluorophores are not available. Red-shifted fluorescent protein (FP) variants would substantially expand the toolkit for spectral unmixing of multiple molecular species, but the naturally occurring red-shifted FPs derived from corals or sea anemones have lower fluorescence quantum yield and inferior photo-stability compared to the avGFP variants. Further manipulation and possible expansion of the chromophore's conjugated system towards the far-red spectral region is also limited by the repertoire of 20 canonical amino acids prescribed by the genetic code. To overcome these limitations, synthetic biology can achieve further spectral red-shifting via insertion of non-canonical amino acids into the chromophore triad. We describe the application of SPI to engineer avGFP variants with novel spectral properties. Protein expression is performed in a tryptophan-auxotrophic E. coli strain and by supplementing growth media with suitable indole precursors. Inside the cells, these precursors are converted to the corresponding tryptophan analogs and incorporated into proteins by the ribosomal machinery in response to UGG codons. The replacement of Trp-66 in the enhanced "cyan" variant of avGFP (ECFP) by an electron-donating 4-aminotryptophan results in GdFP featuring a 108 nm Stokes shift and a strongly red-shifted emission maximum (574 nm), while being thermodynamically more stable than its predecessor ECFP. Residue-specific incorporation of the non-canonical amino acid is analyzed by mass spectrometry. The spectroscopic properties of GdFP are characterized by time-resolved fluorescence spectroscopy as one of the valuable applications of genetically encoded FPs in life sciences.

The characterization of canvas painting by the Serbian artist Milo Milunović using X-ray fluorescence, micro-Raman and FTIR spectroscopy

NASA Astrophysics Data System (ADS)

Damjanović, Lj.; Gajić-Kvaščev, M.; Đurđević, J.; Andrić, V.; Marić-Stojanović, M.; Lazić, T.; Nikolić, S.

2015-10-01

A canvas painting by Milo Milunović "The Inspiration of the poet" was studied by energy dispersive X-Ray fluorescence (EDXRF), micro-Raman and Fourier transform infrared (FTIR) spectroscopy in order to identify materials used by the artist and his painting technique. Study is perfomed combining in situ non-destructive method with the preparation and study of cross-section samples and raw fragments of the samples. Milo Milunović, an eminent painter from Balkan region, made a copy of the Nicolas Poussin's original painting in Louvre in 1926/27. Obtained results revealed following pigments on the investigated canvas painting: vermilion, minium, cobalt blue, ultramarine, lead white, zinc white, cadmium yellow, chrome-based green pigment and several earth pigments - red and yellow ocher, green earth and umber. Ground layer was made of lead white mixed with calcium carbonate.

Noncontact point spectroscopy guided by two-channel fluorescence imaging in a hamster cheek pouch model

NASA Astrophysics Data System (ADS)

Yang, Victor X.; Yeow, Jenny; Lilge, Lothar D.; Kost, James; Mang, Thomas S.; Wilson, Brian C.

1999-07-01

A system for in vivo, fluorescence image-guided, non-contact point fluorescence spectroscopy is presented. A 442 nm HeCd laser is used as the fluorescence excitation source. An intensified CCD serves as the detector for both imaging and spectroscopy, on which two regions of 300 X 300 pixels were used for green (500 +/- 18 nm) and red (630 +/- 18 nm) imaging channels, and a strip of 600 X 120 pixels are used for emission spectroscopy (450 - 750 nm). At a working distance of 40 mm, the system has a spatial resolution of 0.16 mm and a spectral resolution of 5 nm. System performance is demonstrated in a carcinogenesis model in hamsters, where tumors were induced by painting DMBA in the cheek pouch. Autofluorescence and Photofrin-induced fluorescence measurements were performed every 2 weeks during the 18 weeks of tumor induction. Punch biopsies on selected animals were taken for histological staging. The results show that autofluorescence fluorescence can distinguish dysplasia from normal mucosal tissue model, utilizing the peak red intensity (or the red-to-green intensity ratio). Photofrin-induced fluorescence was superior to autofluorescence for differentiating high grade dysplasia from invasive cancer.

Imaging intracellular pH in live cells with a genetically encoded red fluorescent protein sensor.

PubMed

Tantama, Mathew; Hung, Yin Pun; Yellen, Gary

2011-07-06

Intracellular pH affects protein structure and function, and proton gradients underlie the function of organelles such as lysosomes and mitochondria. We engineered a genetically encoded pH sensor by mutagenesis of the red fluorescent protein mKeima, providing a new tool to image intracellular pH in live cells. This sensor, named pHRed, is the first ratiometric, single-protein red fluorescent sensor of pH. Fluorescence emission of pHRed peaks at 610 nm while exhibiting dual excitation peaks at 440 and 585 nm that can be used for ratiometric imaging. The intensity ratio responds with an apparent pK(a) of 6.6 and a >10-fold dynamic range. Furthermore, pHRed has a pH-responsive fluorescence lifetime that changes by ~0.4 ns over physiological pH values and can be monitored with single-wavelength two-photon excitation. After characterizing the sensor, we tested pHRed's ability to monitor intracellular pH by imaging energy-dependent changes in cytosolic and mitochondrial pH.

Fluorescent proteins as biomarkers and biosensors: throwing color lights on molecular and cellular processes.

PubMed

Stepanenko, Olesya V; Verkhusha, Vladislav V; Kuznetsova, Irina M; Uversky, Vladimir N; Turoverov, K K

2008-08-01

Green fluorescent protein (GFP) from jellyfish Aequorea victoria is the most extensively studied and widely used in cell biology protein. GFP-like proteins constitute a fast growing family as several naturally occurring GFP-like proteins have been discovered and enhanced mutants of Aequorea GFP have been created. These mutants differ from wild-type GFP by conformational stability, quantum yield, spectroscopic properties (positions of absorption and fluorescence spectra) and by photochemical properties. GFP-like proteins are very diverse, as they can be not only green, but also blue, orange-red, far-red, cyan, and yellow. They also can have dual-color fluorescence (e.g., green and red) or be non-fluorescent. Some of them possess kindling property, some are photoactivatable, and some are photoswitchable. This review is an attempt to characterize the main color groups of GFP-like proteins, describe their structure and mechanisms of chromophore formation, systemize data on their conformational stability and summarize the main trends of their utilization as markers and biosensors in cell and molecular biology.

Far wing depolarization of light - Generalized absorption profiles. [in laser fluorescence spectroscopy of Sr vapor

NASA Technical Reports Server (NTRS)

Thomann, P.; Burnett, K.; Cooper, J.

1981-01-01

An absorption (and/or emission) event which takes place during a strong collision is called a 'correlated event'. It is discussed how correlated events affect the far red wing depolarization of fluorescence. Attention is given to an atomic vapor which is irradiated by linearly polarized light of a frequency on the red side of the resonance line. Two limiting cases are considered, corresponding to excitation in the impact region and in the quasi-static wing. In the quasi-static wing, absorption of a photon followed by fluorescence (rather than Rayleigh scattering), occurs mostly during a collision. Correlated events dominate the scattering process. Expressions derived for the polarization of the fluorescent light are applied to far red wing depolarization. It is found that the polarization of the fluorescent light does not go to zero in the far wing, but depends crucially on the detailed nature of the anisotropy in the long-range part of the interatomic potential.

Common fluorescent proteins for single-molecule localization microscopy

NASA Astrophysics Data System (ADS)

Klementieva, Natalia V.; Bozhanova, Nina G.; Mishina, Natalie M.; Zagaynova, Elena V.; Lukyanov, Konstantin A.; Mishin, Alexander S.

2015-07-01

Super-resolution techniques for breaking the diffraction barrier are spread out over multiple studies nowadays. Single-molecule localization microscopy such as PALM, STORM, GSDIM, etc allow to get super-resolved images of cell ultrastructure by precise localization of individual fluorescent molecules via their temporal isolation. However, these methods are supposed the use of fluorescent dyes and proteins with special characteristics (photoactivation/photoconversion). At the same time, there is a need for retaining high photostability of fluorophores during long-term acquisition. Here, we first showed the potential of common red fluorescent protein for single-molecule localization microscopy based on spontaneous intrinsic blinking. Also, we assessed the effect of different imaging media on photobleaching of these fluorescent proteins. Monomeric orange and red fluorescent proteins were examined for stochastic switching from a dark state to a bright fluorescent state. We studied fusions with cytoskeletal proteins in NIH/3T3 and HeLa cells. Imaging was performed on the Nikon N-STORM system equipped with EMCCD camera. To define the optimal imaging conditions we tested several types of cell culture media and buffers. As a result, high-resolution images of cytoskeleton structure were obtained. Essentially, low-intensity light was sufficient to initiate the switching of tested red fluorescent protein reducing phototoxicity and provide long-term live-cell imaging.

Fluorescence of the various red antenna states in photosystem I complexes from cyanobacteria is affected differently by the redox state of P700.

PubMed

Schlodder, Eberhard; Hussels, Martin; Cetin, Marianne; Karapetyan, Navassard V; Brecht, Marc

2011-11-01

Photosystem I of cyanobacteria contains different spectral pools of chlorophylls called red or long-wavelength chlorophylls that absorb at longer wavelengths than the primary electron donor P700. We measured the fluorescence spectra at the ensemble and the single-molecule level at low temperatures in the presence of oxidized and reduced P700. In accordance with the literature, it was observed that the fluorescence is quenched by P700(+). However, the efficiency of the fluorescence quenching by oxidized P700(+) was found to be extremely different for the various red states in PS I from different cyanobacteria. The emission of the longest-wavelength absorbing antenna state in PS I trimers from Thermosynechococcus elongatus (absorption maximum at 5K: ≈ 719nm; emission maximum at 5K: ≈ 740nm) was found to be strongly quenched by P700(+) similar to the reddest state in PS I trimers from Arthrospira platensis emitting at 760nm at 5K. The fluorescence of these red states is diminished by more than a factor of 10 in the presence of oxidized P700. For the first time, the emission of the reddest states in A. platensis and T. elongatus has been monitored using single-molecule fluorescence techniques. 2011 Elsevier B.V. All rights reserved.

Highly stable red-emitting polymer dots for cellular imaging

NASA Astrophysics Data System (ADS)

Chelora, Jipsa; Zhang, Jinfeng; Chen, Rui; Thachoth Chandran, Hrisheekesh; Lee, Chun-Sing

2017-07-01

Polymer dots (Pdots) have emerged as a new type of fluorescent probe material for biomedical applications and have attracted great interest due to their excellent optical properties and biocompatability. In this work, we report on a red-emitting P3HT Pdot fluorescent probe for intracellular bioimaging. The as-prepared Pdot fluorescent probe exhibits good stability and has a large Stokes shift (121 nm) compared to molecules in tetrahydrofuran (THF). Furthermore, the probe shows low cytotoxicity, broad absorption spectrum, resistance against photodegradation, and good water dispersibility. These advantageous characteristics make P3HT Pdots a promising fluorescent probe material for bioimaging.

Highly stable red-emitting polymer dots for cellular imaging.

PubMed

Chelora, Jipsa; Zhang, Jinfeng; Chen, Rui; Chandran, Hrisheekesh Thachoth; Lee, Chun-Sing

2017-07-14

Polymer dots (Pdots) have emerged as a new type of fluorescent probe material for biomedical applications and have attracted great interest due to their excellent optical properties and biocompatability. In this work, we report on a red-emitting P 3 HT Pdot fluorescent probe for intracellular bioimaging. The as-prepared Pdot fluorescent probe exhibits good stability and has a large Stokes shift (121 nm) compared to molecules in tetrahydrofuran (THF). Furthermore, the probe shows low cytotoxicity, broad absorption spectrum, resistance against photodegradation, and good water dispersibility. These advantageous characteristics make P 3 HT Pdots a promising fluorescent probe material for bioimaging.

The nature of multiphoton fluorescence from red blood cells

NASA Astrophysics Data System (ADS)

Saytashev, Ilyas; Murphy, Michael; Osseiran, Sam; Spence, Dana M.; Evans, Conor L.; Dantus, Marcos

2016-03-01

We report on the nature of multiphoton excited fluorescence observed from human erythrocytes (red blood cells RBC's) and their "ghosts" following 800nm sub-15 fs excitation. The detected optical signal is assigned as two-photon excited fluorescence from hemoglobin. Our findings are supported by wavelength-resolved fluorescence lifetime decay measurements using time-correlated single photon counting system from RBC's, their ghosts as well as in vitro samples of various fluorophores including riboflavin, NADH, NAD(P)H, hemoglobin. We find that low-energy and short-duration pulses allow two-photon imaging of RBC's, but longer more intense pulses lead to their destruction.

ONIOM Investigation of the Second-Order Nonlinear Optical Responses of Fluorescent Proteins.

PubMed

de Wergifosse, Marc; Botek, Edith; De Meulenaere, Evelien; Clays, Koen; Champagne, Benoît

2018-05-17

The first hyperpolarizability (β) of six fluorescent proteins (FPs), namely, enhanced green fluorescent protein, enhanced yellow fluorescent protein, SHardonnay, ZsYellow, DsRed, and mCherry, has been calculated to unravel the structure-property relationships on their second-order nonlinear optical properties, owing to their potential for multidimensional biomedical imaging. The ONIOM scheme has been employed and several of its refinements have been addressed to incorporate efficiently the effects of the microenvironment on the nonlinear optical responses of the FP chromophore that is embedded in a protective β-barrel protein cage. In the ONIOM scheme, the system is decomposed into several layers (here two) treated at different levels of approximation (method1/method2), from the most elaborated method (method1) for its core (called the high layer) to the most approximate one (method2) for the outer surrounding (called the low layer). We observe that a small high layer can already account for the variations of β as a function of the nature of the FP, provided the low layer is treated at an ab initio level to describe properly the effects of key H-bonds. Then, for semiquantitative reproduction of the experimental values obtained from hyper-Rayleigh scattering experiments, it is necessary to incorporate electron correlation as described at the second-order Møller-Plesset perturbation theory (MP2) level as well as implicit solvent effects accounted for using the polarizable continuum model (PCM). This led us to define the MP2/6-31+G(d):HF/6-31+G(d)/IEFPCM scheme as an efficient ONIOM approach and the MP2/6-31+G(d):HF/6-31G(d)/IEFPCM as a better compromise between accuracy and computational needs. Using these methods, we demonstrate that many parameters play a role on the β response of FPs, including the length of the π-conjugated segment, the variation of the bond length alternation, and the presence of π-stacking interactions. Then, noticing the small diversity of the FP chromophores, these results highlight the key role of the β-barrel and surrounding residues on β, not only because they can locally break the noncentrosymmetry vital to a β response but also because it can impose geometrical constraints on the chromophore.

Spectral fluorescent properties of tissues in vivo with excitation in the red wavelength range

NASA Astrophysics Data System (ADS)

Stratonnikov, Alexander A.; Loschenov, Victor B.; Klimov, D. V.; Edinac, N. E.; Wolnukhin, V. A.; Strashkevich, I. A.

1997-12-01

The spectral fluorescence analysis is a promising method for differential tissue diagnostic. Usually the UV and visible light is used for fluorescence excitation with emission registration in the visible wavelength range. The light penetration length in this wavelength range is very small allowing one to analyze only the surface region of the tissue. Here we present the tissue fluorescent spectra in vivo excited in the red wavelength region. As excitation light source we used compact He-Ne laser (632.8 nm) and observed the fluorescence in 650 - 800 nm spectral range. The various tissues including normal skin, psoriasis, tumors, necrosis as well as photosensitized tissues have been measured.

A far-red fluorescent protein evolved from a cyanobacterial phycobiliprotein.

PubMed

Rodriguez, Erik A; Tran, Geraldine N; Gross, Larry A; Crisp, Jessica L; Shu, Xiaokun; Lin, John Y; Tsien, Roger Y

2016-09-01

Far-red fluorescent proteins (FPs) are desirable for in vivo imaging because with these molecules less light is scattered, absorbed, or re-emitted by endogenous biomolecules compared with cyan, green, yellow, and orange FPs. We developed a new class of FP from an allophycocyanin α-subunit (APCα). Native APC requires a lyase to incorporate phycocyanobilin. The evolved FP, which we named small ultra-red FP (smURFP), covalently attaches a biliverdin (BV) chromophore without a lyase, and has 642/670-nm excitation-emission peaks, a large extinction coefficient (180,000 M(-1)cm(-1)) and quantum yield (18%), and photostability comparable to that of eGFP. smURFP has significantly greater BV incorporation rate and protein stability than the bacteriophytochrome (BPH) FPs. Moreover, BV supply is limited by membrane permeability, and smURFPs (but not BPH FPs) can incorporate a more membrane-permeant BV analog, making smURFP fluorescence comparable to that of FPs from jellyfish or coral. A far-red and near-infrared fluorescent cell cycle indicator was created with smURFP and a BPH FP.

A Passive Method for Detecting Vegetation Stress from Orbit: Chlorophyll Fluorescence Spectra from Fraunhofer Lines

NASA Technical Reports Server (NTRS)

Theisen, Arnold F.

2000-01-01

Solar-stimulated chlorophyll fluorescence measured with the Fraunhofer line depth method has correlated well with vegetation stress in previous studies. However, the instruments used in those studies were limited to a single solar absorption line (e.g. 656.3 nm), obviating the red/far-red ratio (R/FR) method. Optics and detector technology have reached the level whereby multiple, very narrow Fraunhofer lines are resolvable. Thirteen such lines span the visible spectrum in the red to far-red region where chlorophyll fluorescence occurs. Fluorescence intensities at the 13 Fraunhofer line wavelengths were used to model emission spectra. The source data were collected for summer and fall bean crops (Phaseolus vulgaris L.) subjected to various levels of nitrogen fertilization. The intensities were adjusted to account for Fraunhofer line depth and atmospheric transmittance. Multiple R/FR fluorescence ratios, calculated from the modeled fluorescence spectra, correlated strongly with leaf chlorophyll concentration and well with applied nitrogen. The ratio yielding the best correlation with chlorophyll utilized red fluorescence at the 694.5 nm Fraunhofer line and farred fluorescence at the 755.6 nm Fraunhofer line. Twenty R/FR ratios, each evaluated for the maximum differential between low and high (optimal) nitrogen treatments, ranked higher in some cases and lower in others, possibly related to the time of year the crops were grown and the stage of growth of the crops. Ratios with 728.9 nm and 738.9 nm in the denominator consistently ranked in the lowest and next lowest quartile, respectively. Ratios of the 656.3 nm Fraunhofer line and the 755.6 nm line consistently ranked highest for the summer crop. Ratios with 755.6 nm in the denominator ranked in the upper quartile for 10 out of 12 measurement dates. Differences in ratio ranking indicate that physiological conditions may be estimated using selected ratios of Fraunhofer lines within the context of R/FR analysis. A passive instrument designed to monitor R/FR chlorophyll fluorescence (i.e. vegetation stress) from orbit could be built today.

Development of fiber optic spectroscopy for in-vitro and in-planta detection of fluorescent proteins

NASA Astrophysics Data System (ADS)

Liew, Oi Wah; Chen, Jun-Wei; Asundi, Anand K.

2001-10-01

The objective of this project is to apply photonics technology to bio-safety management of genetically modified (GM) plants. The conventional method for screening GM plants is through selection using antibiotic resistance markers. There is public concern with such approaches and these are associated with food safety issues, escape of antibiotic resistance genes to pathogenic microorganisms and interference with antibiotic therapy. Thus, the strategy taken in this project is to replace antibiotic resistance markers with fluorescent protein markers that allow for rapid and non-invasive optical screening of genetically modified plants. In this paper, fibre optic spectroscopy was developed to detect and quantify recombinant green (EGFP) and red (DsRED) fluorescent proteins in vitro and in planta. In vitro detection was first carried out to optimize the sensitivity of the optical system. The bacterial expression vectors carrying the coding regions of EGFP and DsRED were introduced into Escherichia coli host cells and fluorescent proteins were produced following induction with IPTG. Soluble EGFP and DsRED proteins were isolated from lysed bacterial cells and serially diluted for quantitative analysis by fibre optic spectroscopy using different light sources, namely, blue LED (475 nm), tungsten halogen (350 - 1000 nm) and double frequency Nd:YAG green laser (532 nm). Fluorescence near the expected emission wavelengths could be detected up to 320X dilution for EGFP and DsRED with blue LED and 532 nm green laser, respectively, as the excitation source. Tungsten halogen was found to be unsuitable for excitation of both EGFP and DsRED. EGFP was successfully purified by size separation under non-denaturing electrophoretic conditions and quantified. The minimum concentration of EGFP detectable with blue LED excitation was 5 mg/ml. To determine the capability of spectroscopy detection in planta, transgenic potato hairy roots and whole modified plant lines expressing the fluorescent markers were regenerated. T

Spectral cola or how to turn green laser light red

NASA Astrophysics Data System (ADS)

Forró, Csaba; Mettan, Xavier; Humair, Thibaud

2014-11-01

A green laser pointer shone through a glass of cola will appear red when looking from above the glass. Lowering the laser so that it is deeper in the cola, the colour will vary from orange to deep red. Fluorescence and absorption spectra were recorded consistent with the reddening of the laser. The depth dependence of the colour of the immersed laser is attributed to a more significant absorption of the short wavelengths by the cola, making the laser look redder as it is lowered deeper into the cola. To confirm this hypothesis, we simulated the fluorescence spectrum we would get at various depths by applying the Lambert law on our measured fluorescence spectrum. These spectra were then converted into red, green and blue (RGB) values. These values were compared to those measured on images of the immersed laser beam at corresponding depths.

Development of redox-sensitive red fluorescent proteins for imaging redox dynamics in cellular compartments.

PubMed

Fan, Yichong; Ai, Hui-wang

2016-04-01

We recently reported a redox-sensitive red fluorescent protein, rxRFP1, which is one of the first genetically encoded red-fluorescent probes for general redox states in living cells. As individual cellular compartments have different basal redox potentials, we hereby describe a group of rxRFP1 mutants, showing different midpoint redox potentials for detection of redox dynamics in various subcellular domains, such as mitochondria, the cell nucleus, and endoplasmic reticulum (ER). When these redox probes were expressed and subcellularly localized in human embryonic kidney (HEK) 293 T cells, they responded to membrane-permeable oxidants and reductants. In addition, a mitochondrially localized rxRFP1 mutant, Mito-rxRFP1.1, was used to detect mitochondrial oxidative stress induced by doxorubicin-a widely used cancer chemotherapy drug. Our work has expanded the fluorescent protein toolkit with new research tools for studying compartmentalized redox dynamics and oxidative stress under various pathophysiological conditions.

Visualization of Porphyrin-Based Photosensitizer Distribution from Fluorescence Images In Vivo Using an Optimized RGB Camera

NASA Astrophysics Data System (ADS)

Liu, L.; Huang, Zh.; Qiu, Zh.; Li, B.

2018-01-01

A handheld RGB camera was developed to monitor the in vivo distribution of porphyrin-based photosensitizer (PS) hematoporphyrin monomethyl ether (HMME) in blood vessels during photodynamic therapy (PDT). The focal length, f-number, International Standardization Organization (ISO) sensitivity, and shutter speed of the camera were optimized for the solution sample with various HMME concentrations. After the parameter optimization, it was found that the red intensity value of the fluorescence image was linearly related to the fluorescence intensity under investigated conditions. The RGB camera was then used to monitor the in vivo distribution of HMME in blood vessels in a skin-fold window chamber model. The red intensity value of the recorded RGB fluorescence image was found to be linearly correlated to HMME concentrations in the range 0-24 μM. Significant differences in the red to green intensity ratios were observed between the blood vessels and the surrounding tissue.

Red fluorescent protein with reversibly photoswitchable absorbance for photochromic FRET.

PubMed

Subach, Fedor V; Zhang, Lijuan; Gadella, Theodorus W J; Gurskaya, Nadya G; Lukyanov, Konstantin A; Verkhusha, Vladislav V

2010-07-30

We have developed the first red fluorescent protein, named rsTagRFP, which possesses reversibly photoswitchable absorbance spectra. Illumination with blue and yellow light switches rsTagRFP into a red fluorescent state (ON state) or nonfluorescent state (OFF state), respectively. The ON and OFF states exhibit absorbance maxima at 567 and 440 nm, respectively. Due to the photoswitchable absorbance, rsTagRFP can be used as an acceptor for a photochromic Förster resonance energy transfer (pcFRET). The photochromic acceptor facilitates determination of a protein-protein interaction by providing an internal control for FRET. Using pcFRET with EYFP as a donor, we observed an interaction between epidermal growth factor receptor and growth factor receptor-binding protein 2 in live cells by detecting the modulation of both the fluorescence intensity and lifetime of the EYFP donor upon the ON-OFF photoswitching of the rsTagRFP acceptor. 2010 Elsevier Ltd. All rights reserved.

A Naturally Fluorescent Mgp Transgenic Mouse for Angiogenesis and Glaucoma Longitudinal Studies

PubMed Central

Asokan, Priyadarsini; Mitra, Rajendra N.; Periasamy, Ramesh; Han, Zongchao

2018-01-01

Purpose Our goal was to generate and characterize a new mouse model in which only angiogenesis- and glaucoma-relevant tissues would be naturally fluorescent. The Matrix Gla (MGP) gene is highly expressed in vascular smooth muscle cells (VSMC) and trabecular meshwork (TM). We sought to direct our Mgp-Cre.KI mouse recombinase to VSMC/TM cells to produce their longitudinal fluorescent profiles. Methods Homozygous Mgp-Cre.KI mice were crossed with Ai9 homozygous reporter mice harboring a loxP-flanked STOP cassette preventing transcription of a DsRed fluorescent protein (tdTomato). The F1 double-heterozygous (Mgp-tdTomato) was examined by direct fluorescence, whole mount, histology, and fundus photography. Custom-made filters had 554/23 emission and 609/54 exciter nanometer wavelengths. Proof of concept of the model's usefulness was conducted by inducing guided imaging laser burns. Evaluation of a vessel's leakage and proliferation was followed by noninvasive angiography. Results The Mgp-tdTomato mouse was viable, fertile, with normal IOP and ERG. Its phenotype exhibited red paws and snout (cartilage expression), which precluded genotyping. A fluorescent red ring was seen at the limbus and confirmed to be TM expression by histology. The entire retinal vasculature was red fluorescent (VSMC) and directly visualized by fundus photography. Laser burns on the Mgp-tdTomato allowed separation of leakiness and neovascularization evaluation parameters. Conclusions The availability of a transgenic mouse naturally fluorescent in glaucoma-relevant tissues and retinal vasculature brings the unique opportunity to study a wide spectrum of single and combined glaucomatous conditions in vivo. Moreover, the Mgp-tdTomato mouse provides a new tool to study mechanisms and therapeutics of retinal angiogenesis longitudinally. PMID:29392320

A Naturally Fluorescent Mgp Transgenic Mouse for Angiogenesis and Glaucoma Longitudinal Studies.

PubMed

Asokan, Priyadarsini; Mitra, Rajendra N; Periasamy, Ramesh; Han, Zongchao; Borrás, Teresa

2018-02-01

Our goal was to generate and characterize a new mouse model in which only angiogenesis- and glaucoma-relevant tissues would be naturally fluorescent. The Matrix Gla (MGP) gene is highly expressed in vascular smooth muscle cells (VSMC) and trabecular meshwork (TM). We sought to direct our Mgp-Cre.KI mouse recombinase to VSMC/TM cells to produce their longitudinal fluorescent profiles. Homozygous Mgp-Cre.KI mice were crossed with Ai9 homozygous reporter mice harboring a loxP-flanked STOP cassette preventing transcription of a DsRed fluorescent protein (tdTomato). The F1 double-heterozygous (Mgp-tdTomato) was examined by direct fluorescence, whole mount, histology, and fundus photography. Custom-made filters had 554/23 emission and 609/54 exciter nanometer wavelengths. Proof of concept of the model's usefulness was conducted by inducing guided imaging laser burns. Evaluation of a vessel's leakage and proliferation was followed by noninvasive angiography. The Mgp-tdTomato mouse was viable, fertile, with normal IOP and ERG. Its phenotype exhibited red paws and snout (cartilage expression), which precluded genotyping. A fluorescent red ring was seen at the limbus and confirmed to be TM expression by histology. The entire retinal vasculature was red fluorescent (VSMC) and directly visualized by fundus photography. Laser burns on the Mgp-tdTomato allowed separation of leakiness and neovascularization evaluation parameters. The availability of a transgenic mouse naturally fluorescent in glaucoma-relevant tissues and retinal vasculature brings the unique opportunity to study a wide spectrum of single and combined glaucomatous conditions in vivo. Moreover, the Mgp-tdTomato mouse provides a new tool to study mechanisms and therapeutics of retinal angiogenesis longitudinally.

zFP538, a yellow fluorescent protein from coral, belongs to the DsRed subfamily of GFP-like proteins but possesses the unexpected site of fragmentation.

PubMed

Zagranichny, Vasily E; Rudenko, Natalia V; Gorokhovatsky, Andrey Yu; Zakharov, Mikhail V; Shenkarev, Zakhar O; Balashova, Tamara A; Arseniev, Alexander S

2004-04-27

The yellow fluorescent protein (zFP538) from coral Zoanthus sp. belongs to a family of green fluorescent protein (GFP). Absorption and emission spectra of zFP538 show an intermediate bathochromic shift as compared with a number of recently cloned GFP-like red fluorescent and nonfluorescent chromoproteins of the DsRed subfamily. Here we report that the zFP538 chromophore is very close, if not identical, in chemical structure to that of DsRed. To gain insight into the mechanism of zFP538 fluorescence and chromophore structure and chemistry, we studied three chromophore-containing peptides isolated from enzymatic digests of zFP538. Like GFP and DsRed chromophores, these contain a p-hydroxybenzylideneimidazolinone moiety formed by Lys-66, Tyr-67, and Gly-68 of zFP538. One of the peptides studied, the hexapeptide FKYGDR derivative, is a proteolysis product of the zFP538 full-length polypeptide containing a GFP-type chromophore already formed and arrested at an earlier stage of maturation. The two other peptides are the derivatives of the pentapeptide KYGDR resulted from the protein in which the chromophore maturation process had been completed. One of these has an oxogroup at Lys-66 C(alpha) and is a hydrolysis product of another one, with the imino group at Lys-66 C(alpha). The N-unsubstituted imino moiety of the latter is generated by spontaneous polypeptide chain fragmentation at a very unexpected site, the former peptide bond between Phe-65 C' and Lys-66 N(alpha). Also observed in the entire protein under mild denaturing conditions, this fragmentation is likely the feature of native zFP538 chromophore that distinguishes it chemically from the DsRed chromophore.

Hydrogen bond strengthening induces fluorescence quenching of PRODAN derivative by turning on twisted intramolecular charge transfer

NASA Astrophysics Data System (ADS)

Yang, Yonggang; Li, Donglin; Li, Chaozheng; Liu, YuFang; Jiang, Kai

2017-12-01

Researchers have proposed different effective mechanisms of hydrogen bonding (HB) on the fluorescence of 6-propionyl-2-dimethylaminonaphthalene (PRODAN) and its derivatives. Herein, excited state transition and dynamics analysis confirm that the fluorescence of PD (a derivative of PRODAN with ethyl replaced by 3-hydroxy-2,2-dimethylpropan) emits from the planar intramolecular charge transfer (PICT) state rather than twist ICT (TICT) state, because the fluorescence emission and surface hopping from the TICT state to the twist ground (T-S0) state is energy forbidden. Nevertheless, the strengthening of intramolecular-HB (intra-HB) and intermolecular-HB (inter-HB) of PD-(methanol)2 smooth the pathway of surface hopping from TICT to T-S0 state and the external conversion going to planar ground state by decreasing the energy difference of the two states. This smoothing changes the fluorescence state of PD-(methanol)2 to the TICT state in which fluorescence emission does not occur but surface hopping, leading to the partial fluorescence quenching of PD in methanol solvent. This conclusion is different from previous related reports. Moreover, the inter-HB strengthening of PD-methanol in PICT state induces the cleavage of intra-HB and a fluorescence red-shift of 54 nm compared to PD. This red-shift increases to 66 nm for PD-(methanol)2 for the strengthening of the one intra-HB and two inter-HBs. The dipole moments of PD-methanol and PD-(methanol)2 respectively increase about 10.3D and 8.1D in PICT state compared to PD. The synergistic effect of intra-HB and inter-HB induces partial quenching of PD in methanol solvent by turning on the TICT state and fluorescence red-shift. This work gives a reasonable description on the fluorescence red-shift and partial quenching of PD in methanol solvent, which will bring insight into the study of spectroscopic properties of molecules owning better spectral characteristics.

Hydrogen bond strengthening induces fluorescence quenching of PRODAN derivative by turning on twisted intramolecular charge transfer.

PubMed

Yang, Yonggang; Li, Donglin; Li, Chaozheng; Liu, YuFang; Jiang, Kai

2017-12-05

Researchers have proposed different effective mechanisms of hydrogen bonding (HB) on the fluorescence of 6-propionyl-2-dimethylaminonaphthalene (PRODAN) and its derivatives. Herein, excited state transition and dynamics analysis confirm that the fluorescence of PD (a derivative of PRODAN with ethyl replaced by 3-hydroxy-2,2-dimethylpropan) emits from the planar intramolecular charge transfer (PICT) state rather than twist ICT (TICT) state, because the fluorescence emission and surface hopping from the TICT state to the twist ground (T-S 0 ) state is energy forbidden. Nevertheless, the strengthening of intramolecular-HB (intra-HB) and intermolecular-HB (inter-HB) of PD-(methanol) 2 smooth the pathway of surface hopping from TICT to T-S 0 state and the external conversion going to planar ground state by decreasing the energy difference of the two states. This smoothing changes the fluorescence state of PD-(methanol) 2 to the TICT state in which fluorescence emission does not occur but surface hopping, leading to the partial fluorescence quenching of PD in methanol solvent. This conclusion is different from previous related reports. Moreover, the inter-HB strengthening of PD-methanol in PICT state induces the cleavage of intra-HB and a fluorescence red-shift of 54nm compared to PD. This red-shift increases to 66nm for PD-(methanol) 2 for the strengthening of the one intra-HB and two inter-HBs. The dipole moments of PD-methanol and PD-(methanol) 2 respectively increase about 10.3D and 8.1D in PICT state compared to PD. The synergistic effect of intra-HB and inter-HB induces partial quenching of PD in methanol solvent by turning on the TICT state and fluorescence red-shift. This work gives a reasonable description on the fluorescence red-shift and partial quenching of PD in methanol solvent, which will bring insight into the study of spectroscopic properties of molecules owning better spectral characteristics. Copyright © 2017 Elsevier B.V. All rights reserved.

A facile synthesis of strong near infrared fluorescent layered double hydroxide nanovehicles with an anticancer drug for tumor optical imaging and therapy

NASA Astrophysics Data System (ADS)

Chen, Chunping; Yee, Lee Kim; Gong, Hua; Zhang, Yong; Xu, Rong

2013-05-01

In this work, a new multifunctional nanovehicle for tumor optical imaging and therapy was developed using Y2O3:Er3+,Yb3+ nanoparticles as near infrared fluorescent nanophosphors, and MgAl-layered double hydroxide (LDH) nanosheets as anticancer drug nanovehicles. Monodispersed Y2O3:Er3+,Yb3+ nanophosphors were readily synthesized by the urea assisted homogenous precipitation method. Hierarchically structured LDH nanosheets intercalated with an anticancer drug, fluorouracil (5FU), were deposited on the surface of Y2O3:Er3+,Yb3+@SiO2 by a simple precipitation method followed by hydrothermal treatment. The resultant Y2O3:Er3+,Yb3+@SiO2@LDH-5FU nanovehicles exhibit strong red upconversion fluorescence under the excitation of a 980 nm laser, which allows tracking of the nanovehicles after localization in cancer cells. A better anticancer efficiency was obtained over the nanovehicles than the free drug which can be attributed to their positively charged surfaces for favorable interaction with the negatively charged cell membranes. The multifunctional nanovehicles designed in this work are expected to be promising material candidates for simultaneous tumor optical imaging and therapy.In this work, a new multifunctional nanovehicle for tumor optical imaging and therapy was developed using Y2O3:Er3+,Yb3+ nanoparticles as near infrared fluorescent nanophosphors, and MgAl-layered double hydroxide (LDH) nanosheets as anticancer drug nanovehicles. Monodispersed Y2O3:Er3+,Yb3+ nanophosphors were readily synthesized by the urea assisted homogenous precipitation method. Hierarchically structured LDH nanosheets intercalated with an anticancer drug, fluorouracil (5FU), were deposited on the surface of Y2O3:Er3+,Yb3+@SiO2 by a simple precipitation method followed by hydrothermal treatment. The resultant Y2O3:Er3+,Yb3+@SiO2@LDH-5FU nanovehicles exhibit strong red upconversion fluorescence under the excitation of a 980 nm laser, which allows tracking of the nanovehicles after localization in cancer cells. A better anticancer efficiency was obtained over the nanovehicles than the free drug which can be attributed to their positively charged surfaces for favorable interaction with the negatively charged cell membranes. The multifunctional nanovehicles designed in this work are expected to be promising material candidates for simultaneous tumor optical imaging and therapy. Electronic supplementary information (ESI) available: TEM images of Y2O3:Er3+,Yb3+@SiO2 synthesized by using different amounts of TEOS, and confocal scanning laser microscopy images (Z stack) of MCF-7 cells incubated with Y2O3:Er3+,Yb3+@SiO2@LDH-5FU for 30 min and 24 h. See DOI: 10.1039/c3nr00781b

LysoTracker and MitoTracker Red are transport substrates of P-glycoprotein: implications for anticancer drug design evading multidrug resistance.

PubMed

Zhitomirsky, Benny; Farber, Hodaya; Assaraf, Yehuda G

2018-04-01

LysoTracker and MitoTracker Red are fluorescent probes widely used for viable cell staining of lysosomes and mitochondria, respectively. They are utilized to study organelle localization and their resident proteins, assess organelle functionality and quantification of organelle numbers. The ATP-driven efflux transporter P-glycoprotein (P-gp) is expressed in normal and malignant tissues and extrudes structurally distinct endogenous and exogenous cytotoxic compounds. Thus, once aromatic hydrophobic compounds such as the above-mentioned fluorescent probes are recognized as transport substrates, efflux pumps including P-gp may abolish their ability to reach their cellular target organelles. Herein, we show that LysoTracker and MitoTracker Red are expelled from P-gp-overexpressing cancer cells, thus hindering their ability to fluorescently mark target organelles. We further demonstrate that tariquidar, a potent P-gp transport inhibitor, restores LysoTracker and MitoTracker Red cell entry. We conclude that LysoTracker and MitoTracker Red are P-gp transport substrates, and therefore, P-gp expression must be taken into consideration prior to cellular applications using these probes. Importantly, as MitoTracker was a superior P-gp substrate than LysoTracker Red, we discuss the implications for the future design of chemotherapeutics evading cancer multidrug resistance. Furthermore, restoration of MitoTracker Red fluorescence in P-gp-overexpressing cells may facilitate the identification of potent P-gp transport inhibitors (i.e. chemosensitizers). © 2018 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.

Integrated Fluorescence

NASA Technical Reports Server (NTRS)

Tuma, Margaret (Inventor); Gruhlke, Russell W. (Inventor)

1998-01-01

A detection method is integrated with a filtering method and an enhancement method to create a fluorescence sensor that can be miniaturized. The fluorescence sensor comprises a thin film geometry including a waveguide layer, a metal film layer and sensor layer. The thin film geometry of the fluorescence sensor allows the detection of fluorescent radiation over a narrow wavelength interval. This enables wavelength discrimination and eliminates the detection of unwanted light from unknown or spurious sources.

Chromatic shifts in the fluorescence emitted by murine thymocytes stained with Hoechst 33342.

PubMed

Petersen, Timothy W; Ibrahim, Sherrif F; Diercks, Alan H; van den Engh, Ger

2004-08-01

Many methods in flow cytometry rely on staining DNA with a fluorescent dye to gauge DNA content. From the relative intensity of the fluorescence signature, one can then infer position in cell cycle, amount of DNA (i.e., for sperm selection), or, as in the case of flow karyotyping, to distinguish individual chromosomes. This work examines the staining of murine thymocytes with a common DNA dye, Hoechst 33342, to investigate nonlinearities in the florescence intensity as well as chromatic shifts. Murine thymocytes were stained with Hoechst 33342 and measured in a flow cytometer at two fluorescence emission bands. In other measurements, cells were stained at different dye concentrations, and then centrifuged. The supernatant was then used for a second round of staining to test the amount of dye uptake. Finally, to test for resonant energy transfer, we measured fluorescence anisotropy at two different wavelengths. The fluorescence of cells stained with Hoechst 33342 is a nonlinear process that shows an overall decrease in intensity with increased dye uptake, and spectral shift to the red. Along with the spectral shift of the fluorescence to the longer wavelengths, we document decreases in the fluorescence anisotropy that may indicate resonant energy transfer. At low concentrations, Hoechst 33342 binds to the minor groove of DNA and shows an increase in fluorescence and a blue shift upon binding. At higher concentrations, at which the dye molecules can no longer bind without overlapping, the blue fluorescence decreases and the red fluorescence increases until there is approximately one dye molecule per DNA base pair. The ratio of the blue fluorescence to the red fluorescence is an accurate indicator of the cellular dye concentration.

Protoporphyrin IX fluorescence as potential indicator of psoriasis severity and progression.

PubMed

Wang, Bo; Xu, Yu-Ting; Zhang, Li; Zheng, Jie; Sroka, Ronald; Wang, Hong-Wei; Wang, Xiu-Li

2017-09-01

In psoriatic lesions, fluorescence diagnosis with blue light can detect protoporphyrin IX accumulation, especially after topical 5-aminolaevulinic acid (ALA) application. However, variable fluorescence distributions, interpersonal variations and long incubation time limit its wide application in clinic. This study is aimed to identify a consistent and convenient method to facilitate diagnosis and evaluation of psoriatic lesions. 104 psoriatic lesions from 30 patients were evaluated. Single lesion PSI scoring and fluorescence by macrospectrofluorometry were recorded on each lesion before and after treatment with narrow-band UVB. Punctate red fluorescence, emitted mainly by protoporphyrin IX, is observed in some psoriatic lesions. According to psoriasis severity index, fluorescence-positive lesions are more severe than lesions without fluorescence. We found a significant positive correlation between psoriasis severity and fluorescence intensity from protoporphyrin IX. Protoporphyrin IX-induced red fluorescence can be used as a novel and convenient approach for psoriasis diagnosis and progression evaluation. Copyright © 2017. Published by Elsevier B.V.

Application of concentrating plasmonic luminescent down-shifting layers for photovoltaic devices

NASA Astrophysics Data System (ADS)

Ahmed, H.; Rafiee, M.; Chandra, S.; Sethi, A.; McCormack, S. J.

2017-02-01

In this paper, concentrating structures of plasmonic luminescent downshifting composite layers (c-pLDS) containing lumogen yellow dye and silver nanoparticles (Ag NPs) to increase the efficiency of Photovoltaic (PV) devices were investigated. The c-pLDS structures allowed for a wider absorption range of both wavelength shifting and light concentration with a strong energy transfer that red shifts photons to wavelengths which gives greater spectral response of solar cells. The optimum dye concentration in a poly(methyl,methacrylate) polymer of a thin layer 10μm spin coated on glass substrate was established. Subsequently, plasmonic coupling with Ag NPs was introduced for the c-pLDS composite structures. Plasmonic coupling has been observed to produce fluorescence emission enhancement of up to 20% for the dye c-pLDS layer. The c-pLDS layer was modelled for CdTe mini modules (15x15 cm) and compared with a blank PMMA/GLASS and dye c-LDS structure. It has been demonstrated that the addition of c-pLDS layers containing lumogen yellow dye increases the optical efficiency and the Short circuit current (Jsc) of CdTe solar cells. An increase of 7.3% in the optical efficiency has been achieved and a 30% in the Jsc was obtained when a c-pLDS composite layer is used.

A Laboratory Exercise for Visible Gel Filtration Chromatography Using Fluorescent Proteins

ERIC Educational Resources Information Center

Zhang, Wenqiang; Cao, Yibin; Xu, Lishan; Gong, Jufang; Sun, Meihao

2015-01-01

Gel filtration chromatography (GFC) separates molecules according to size and is one of the most widely used methods for protein purification. Here, red fluorescent protein (RFP), green fluorescent protein (GFP), yellow fluorescent protein (YFP), cyan fluorescent protein (CFP), and/or their fusion proteins were prokaryotically expressed, purified,…

Detection of Silver Nanoparticles in Cells by Flow Cytometry Using Light Scattering and Far-red Fluorescence

EPA Science Inventory

The cellular uptake of different sized silver nanoparticles (l0 nm, 50 nm, and 75nm) coated with polyvinylpyrrolidone (PVP) or citrate in ARPE-19 cells following 24 hour incubation was detected by side scatter through the use of a flow cytometer. A large far red fluorescence sign...

The Radiative Decay of Green and Red Photoluminescent Phosphors: An Undergraduate Kinetics Experiment for Materials Chemistry

ERIC Educational Resources Information Center

Esposti, C. Degli; Bizzocchi, L.

2008-01-01

This article describes a laboratory experiment that allows the students to investigate the radiative properties of the green and red emitting phosphors that are employed in commercial fluorescent lamps. Making use of a spectrofluorometer, students first record the emission spectrum of a fluorescent lamp under normal operating conditions, and then…

Daily light use efficiency in a cornfield can be related to the canopy red/far-red fluorescence ratio and leaf light use efficiency across a growing season

USDA-ARS?s Scientific Manuscript database

In multiple years (2008-2013), we collected canopy and leaf fluorescence, photosynthesis, hyperspectral reflectance spectra, and biophysical measurements along transects within a USDA/Beltsville experimental cornfield treated with optimal nitrogen application (100%N) and which has an eddy covariance...

Real-time detection of caspase-2 activation in a single living HeLa cell during cisplatin-induced apoptosis

NASA Astrophysics Data System (ADS)

Lin, Juqiang; Zhang, Zhihong; Yang, Jie; Zeng, Shaoqun; Liu, Bifeng; Luo, Qingming

2006-03-01

Caspase-2 is important for the mitochondrial apoptotic pathway, however, the mechanism by which caspase-2 executes apoptosis remains obscure. We carry out the first measurements of the dynamics of caspase-2 activation in a single living cell by a FRET (fluorescence resonance energy transfer) probe. Two FRET probes are constructed that each encoded a CRS (caspase-2 or caspase-3 recognition site) fused with a cyan fluorescent protein (CFP) and a red fluorescent protein (DsRed) (CFP-CRS-DsRed). Using these probes, we found that during cisplatin-induced apoptosis, caspase-2 activation occurred more slowly than did activation of caspase-3; additionally, caspase-2 activation was initiated much earlier than that of caspase-3.

α-MHC MitoTimer mouse: In vivo mitochondrial turnover model reveals remarkable mitochondrial heterogeneity in the heart

PubMed Central

Stotland, Aleksandr; Gottlieb, Roberta A.

2016-01-01

In order to maintain an efficient, energy-producing network in the heart, dysfunctional mitochondria are cleared through the mechanism of autophagy, which is closely linked with mitochondrial biogenesis; these, together with fusion and fission comprise a crucial process known as mitochondrial turnover. Until recently, the lack of molecular tools and methods available to researchers has impeded in vivo investigations of turnover. To investigate the process at the level of a single mitochondrion, our laboratory has developed the MitoTimer protein. Timer is a mutant of DsRed fluorescent protein characterized by transition from green fluorescence to a more stable red conformation over 48 h, and its rate of maturation is stable under physiological conditions. We fused the Timer cDNA with the inner mitochondrial membrane signal sequence and placed it under the control of a cardiac-restricted promoter. This construct was used to create the alpha-MHC-MitoTimer mice. Surprisingly, initial analysis of the hearts from these mice demonstrated a high degree of heterogeneity in the ratio of red-to-green fluorescence of MitoTimer in cardiac tissue. Further, scattered solitary mitochondria within cardiomyocytes display a much higher red-to-green fluorescence (red-shifted) relative to other mitochondria in the cell, implying a block in import of newly synthesized MitoTimer likely due to lower membrane potential. These red-shifted mitochondria may represent older, senescent mitochondria. Concurrently, the cardiomyocytes also contain a subpopulation of mitochondria that display a lower red-to-green fluorescence (green-shifted) relative to other mitochondria, indicative of germinal mitochondria that are actively engaged in import of newly-synthesized mito-targeted proteins. These mitochondria can be isolated and sorted from the heart by flow cytometry for further analysis. Initial studies suggest that these mice represent an elegant tool for the investigation of mitochondrial turnover in the heart. PMID:26654779

Enhanced fluorescence detection using liquid-liquid extraction in a microfluidic droplet system.

PubMed

Chen, Yan-Yu; Chen, Zhao-Ming; Wang, Hsiang-Yu

2012-11-07

Reducing the fluorescence background in microfluidic assays is important in obtaining accurate outcomes and enhancing the quality of detections. This study demonstrates an integrated process including cell labelling, fluorescence background reduction, and biomolecule detection using liquid-liquid extraction in a microfluidic droplet system. The cellular lipids in Chlorella vulgaris and NIH/3T3 cells were labelled with a hydrophobic dye, Nile red, to investigate the performance of the proposed method. The fluorescence background of the lipid detection can be reduced by 85% and the removal efficiency increased with the volume of continuous phase surrounding a droplet. The removal rate of the fluorescence background increased as the surface area to volume ratio of a droplet increased. Before Nile red was removed from the droplet, the signal to noise ratio was as low as 1.30 and it was difficult to distinguish cells from the background. Removing Nile red increased the signal to noise ratio to 22 and 34 for Chlorella vulgaris and NIH/3T3, respectively, and these were 17 fold and 10 fold of the values before extraction. The proposed method successfully demonstrates the enhancement of fluorescence detection of cellular lipids and has great potential in improving other fluorescence-based detections in microfluidic systems.

Canopy Level Chlorophyll Fluorescence and the PRI in a Cornfield

NASA Technical Reports Server (NTRS)

Middleton, Elizabeth M.; Cheng, Yen-Ben; Corp, Lawrence A.; Campbell, Petya K. E.; Huemmrich, K. Fred; Zhang, Qingyuan; Kustas, William P.

2012-01-01

Two bio-indicators, the Photochemical Reflectance Index (PRI) and solar-induced red and far-red Chlorophyll Fluorescence (SIF), were derived from directional hyperspectral observations and studied in a cornfield on two contrasting days in the growing season. Both red and far-red SIF exhibited higher values on the day when the canopy in the early senescent stage, but only the far-red SIF showed sensitivity to viewing geometry. Consequently, the red/far-red SIF ratio varied greatly among azimuth positions while the largest values were obtained for the "hotspot" at both growth stages. This ratio was lower (approx.0.88 +/- 0.4) in early July than in August when the ratio approached equivalence (near approx.1). In concert, the PRI exhibited stronger responses to both zenith and azimuth angles and different values on the two growth stages. The potential of using these indices to monitor photosynthetic activities needs further investigation

Multimodal, 3D pathology-mimicking bladder phantom for evaluation of cystoscopic technologies (Conference Presentation)

NASA Astrophysics Data System (ADS)

Smith, Gennifer T.; Lurie, Kristen L.; Zlatev, Dimitar V.; Liao, Joseph C.; Ellerbee, Audrey K.

2016-02-01

Optical coherence tomography (OCT) and blue light cystoscopy (BLC) have shown significant potential as complementary technologies to traditional white light cystoscopy (WLC) for early bladder cancer detection. Three-dimensional (3D) organ-mimicking phantoms provide realistic imaging environments for testing new technology designs, the diagnostic potential of systems, and novel image processing algorithms prior to validation in real tissue. Importantly, the phantom should mimic features of healthy and diseased tissue as they appear under WLC, BLC, and OCT, which are sensitive to tissue color and structure, fluorescent contrast, and optical scattering of subsurface layers, respectively. We present a phantom posing the hollow shape of the bladder and fabricated using a combination of 3D-printing and spray-coating with Dragon Skin (DS) (Smooth-On Inc.), a highly elastic polymer to mimic the layered structure of the bladder. Optical scattering of DS was tuned by addition of titanium dioxide, resulting in scattering coefficients sufficient to cover the human bladder range (0.49 to 2.0 mm^-1). Mucosal vasculature and tissue coloration were mimicked with elastic cord and red dye, respectively. Urethral access was provided through a small hole excised from the base of the phantom. Inserted features of bladder pathology included altered tissue color (WLC), fluorescence emission (BLC), and variations in layered structure (OCT). The phantom surface and underlying material were assessed on the basis of elasticity, optical scattering, layer thicknesses, and qualitative image appearance. WLC, BLC, and OCT images of normal and cancerous features in the phantom qualitatively matched corresponding images from human bladders.

pHuji, a pH-sensitive red fluorescent protein for imaging of exo- and endocytosis

PubMed Central

Shen, Yi; Rosendale, Morgane

2014-01-01

Fluorescent proteins with pH-sensitive fluorescence are valuable tools for the imaging of exocytosis and endocytosis. The Aequorea green fluorescent protein mutant superecliptic pHluorin (SEP) is particularly well suited to these applications. Here we describe pHuji, a red fluorescent protein with a pH sensitivity that approaches that of SEP, making it amenable for detection of single exocytosis and endocytosis events. To demonstrate the utility of the pHuji plus SEP pair, we perform simultaneous two-color imaging of clathrin-mediated internalization of both the transferrin receptor and the β2 adrenergic receptor. These experiments reveal that the two receptors are differentially sorted at the time of endocytic vesicle formation. PMID:25385186

Multispectral laser-induced fluorescence imaging system for large biological samples

NASA Astrophysics Data System (ADS)

Kim, Moon S.; Lefcourt, Alan M.; Chen, Yud-Ren

2003-07-01

A laser-induced fluorescence imaging system developed to capture multispectral fluorescence emission images simultaneously from a relatively large target object is described. With an expanded, 355-nm Nd:YAG laser as the excitation source, the system captures fluorescence emission images in the blue, green, red, and far-red regions of the spectrum centered at 450, 550, 678, and 730 nm, respectively, from a 30-cm-diameter target area in ambient light. Images of apples and of pork meat artificially contaminated with diluted animal feces have demonstrated the versatility of fluorescence imaging techniques for potential applications in food safety inspection. Regions of contamination, including sites that were not readily visible to the human eye, could easily be identified from the images.

Transgenic nude mouse with green fluorescent protein expression-based human glioblastoma multiforme animal model with EGFR expression and invasiveness.

PubMed

Tan, Guo-Wei; Lan, Fo-Lin; Gao, Jian-Guo; Jiang, Cai-Mou; Zhang, Yi; Huang, Xiao-Hong; Ma, Yue-Hong; Shao, He-Dui; He, Xue-Yang; Chen, Jin-Long; Long, Jian-Wu; Xiao, Hui-Sheng; Guo, Zhi-Tong; Diao, Yi

2012-08-01

Previously, we developed an orthotopic xenograft model of human glioblastoma multiforme (GBM) with high EGFR expression and invasiveness in Balb/c nu/nu nude mice. Now we also developed the same orthotopic xenograft model in transgenic nude mice with green fluorescent protein (GFP) expression. The present orthotopic xenografts labeled by phycoerythrin fluorescing red showed high EGFR expression profile, and invasive behavior under a bright green-red dual-color fluorescence background. A striking advantage in the present human GBM model is that the change of tumor growth can be observed visually instead of sacrificing animals in our further antitumor therapy studies.

[Ph-Sensor Properties of a Fluorescent Protein from Dendronephthya sp].

PubMed

Pakhomov, A A; Chertkova, R V; Martynov, V I

2015-01-01

Genetically encoded biosensors based on fluorescent proteins are now widely applicable for monitoring pH changes in live cells. Here, we have shown that a fluorescent protein from Dendronephthya sp. (DendFP) exhibits a pronounced pH-sensitivity. Unlike most of known genetically encoded pH-sensors, fluorescence of the protein is not quenched upon medium acidification, but is shifting from the red to green spectral range. Therefore, quantitative measurements of intracellular pH are feasible by ratiometric comparison of emission intensities in the red and green spectral ranges, which makes DendFP advantageous compared with other genetically encoded pH-sensors.

Release of Reactive Oxygen Intermediates (Superoxide Radicals, Hydrogen Peroxide, and Hydroxyl Radicals) and Peroxidase in Germinating Radish Seeds Controlled by Light, Gibberellin, and Abscisic Acid1

PubMed Central

Schopfer, Peter; Plachy, Claudia; Frahry, Gitta

2001-01-01

Germination of radish (Raphanus sativus cv Eterna) seeds can be inhibited by far-red light (high-irradiance reaction of phytochrome) or abscisic acid (ABA). Gibberellic acid (GA3) restores full germination under far-red light. This experimental system was used to investigate the release of reactive oxygen intermediates (ROI) by seed coats and embryos during germination, utilizing the apoplastic oxidation of 2′,7′-dichlorofluorescin to fluorescent 2′,7′-dichlorofluorescein as an in vivo assay. Germination in darkness is accompanied by a steep rise in ROI release originating from the seed coat (living aleurone layer) as well as the embryo. At the same time as the inhibition of germination, far-red light and ABA inhibit ROI release in both seed parts and GA3 reverses this inhibition when initiating germination under far-red light. During the later stage of germination the seed coat also releases peroxidase with a time course affected by far-red light, ABA, and GA3. The participation of superoxide radicals, hydrogen peroxide, and hydroxyl radicals in ROI metabolism was demonstrated with specific in vivo assays. ROI production by germinating seeds represents an active, developmentally controlled physiological function, presumably for protecting the emerging seedling against attack by pathogens. PMID:11299341

Fluorescent protein Dendra2 as a ratiometric genetically encoded pH-sensor.

PubMed

Pakhomov, Alexey A; Martynov, Vladimir I; Orsa, Alexander N; Bondarenko, Alena A; Chertkova, Rita V; Lukyanov, Konstantin A; Petrenko, Alexander G; Deyev, Igor E

2017-12-02

Fluorescent protein Dendra2 is a monomeric GFP-like protein that belongs to the group of Kaede-like photoconvertible fluorescent proteins with irreversible photoconversion from a green- to red-emitting state when exposed to violet-blue light. In an acidic environment, photoconverted Dendra2 turns green due to protonation of the phenolic group of the chromophore with pKa of about 7.5. Thus, photoconverted form of Dendra2 can be potentially used as a ratiometric pH-sensor in the physiological pH range. However, incomplete photoconversion makes ratiometric measurements irreproducible when using standard filter sets. Here, we describe the method to detect fluorescence of only photoconverted Dendra2 form, but not nonconverted green Dendra2. We show that the 350 nm excitation light induces solely the fluorescence of photoconverted protein. By measuring the red to green fluorescence ratio, we determined intracellular pH in live CHO and HEK 293 cells. Thus, Dendra2 can be used as a novel ratiometric genetically encoded pH sensor with emission maxima in the green-red spectral region, which is suitable for application in live cells. Copyright © 2017 Elsevier Inc. All rights reserved.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Pletneva, Nadya V.; Pletnev, Vladimir Z.; Shemiakina, Irina I.

The wild type red fluorescent protein eqFP578 (from sea anemone Entacmaea quadricolor, {lambda}{sub ex} = 552 nm, {lambda}{sub em} = 578 nm) and its bright far-red fluorescent variant Katushka ({lambda}{sub ex} = 588 nm, {lambda}{sub em} = 635 nm) are characterized by the pronounced pH dependence of their fluorescence. The crystal structures of eqFP578f (eqFP578 with two point mutations improving the protein folding) and Katushka have been determined at the resolution ranging from 1.15 to 1.85 {angstrom} at two pH values, corresponding to low and high level of fluorescence. The observed extinguishing of fluorescence upon reducing pH in eqFP578f andmore » Katushka has been shown to be accompanied by the opposite trans-cis and cis-trans chromophore isomerization, respectively. Asn143, Ser158, His197 and Ser143, Leu174, and Arg197 have been shown to stabilize the respective trans and cis fluorescent states of the chromophores in eqFP578f and Katushka at higher pH. The cis state has been suggested as being primarily responsible for the observed far-red shift of the emission maximum of Katushka relative to that of eqFP578f.« less

Fluorescent measurements in whole blood and plasma using red-emitting dyes

NASA Astrophysics Data System (ADS)

Abugo, Omoefe O.; Herman, Petr; Lakowicz, Joseph R.

2000-04-01

We have determined the fluorescence characteristics of albumin blue 670 and Rhodamine 800 in plasma and blood in order to test the feasibility of making direct fluorescence sensing measurements in blood. These dyes were used because of their absorption in the red/NIR where absorption by hemoglobin is minimized. Front face illumination and detection was used to minimize absorption and scattering during measurement. Fluorescence emission was observed for these dyes in plasma and blood. Attenuation of the fluorescence emission was observed in blood because of hemoglobin absorption. Using frequency domain fluorometry, we recovered the expected lifetime parameters for both dyes in blood and plasma. We were able to quantify HSA concentrations using changes in the mean lifetime of AB670, a dye previously shown to bind preferentially to HSA. Rh800 concentrations in plasma and blood were also determined using modulation sensing. Anisotropy measurements revealed high Anisotropy for these dyes in plasma and blood. It also showed an increase in the anisotropy of AB670 with increase in HSA concentration in the presence of red blood cells. These results indicate that qualitative and quantitative fluorescence measurements can be made directly in blood without the need to process the blood.

Green-light supplementation for enhanced lettuce growth under red- and blue-light-emitting diodes

NASA Technical Reports Server (NTRS)

Kim, Hyeon-Hye; Goins, Gregory D.; Wheeler, Raymond M.; Sager, John C.

2004-01-01

Plants will be an important component of future long-term space missions. Lighting systems for growing plants will need to be lightweight, reliable, and durable, and light-emitting diodes (LEDs) have these characteristics. Previous studies demonstrated that the combination of red and blue light was an effective light source for several crops. Yet the appearance of plants under red and blue lighting is purplish gray making visual assessment of any problems difficult. The addition of green light would make the plant leave appear green and normal similar to a natural setting under white light and may also offer a psychological benefit to the crew. Green supplemental lighting could also offer benefits, since green light can better penetrate the plant canopy and potentially increase plant growth by increasing photosynthesis from the leaves in the lower canopy. In this study, four light sources were tested: 1) red and blue LEDs (RB), 2) red and blue LEDs with green fluorescent lamps (RGB), 3) green fluorescent lamps (GF), and 4) cool-white fluorescent lamps (CWF), that provided 0%, 24%, 86%, and 51% of the total PPF in the green region of the spectrum, respectively. The addition of 24% green light (500 to 600 nm) to red and blue LEDs (RGB treatment) enhanced plant growth. The RGB treatment plants produced more biomass than the plants grown under the cool-white fluorescent lamps (CWF treatment), a commonly tested light source used as a broad-spectrum control.

Light-energy processing and freezing-tolerance traits in red spruce and black spruce: species and seed-source variation.

PubMed

Major, John E; Barsi, Debby C; Mosseler, Alex; Campbell, Moira; Rajora, Om P

2003-07-01

Red spruce (Picea rubens Sarg.) and black spruce (Picea mariana (Mill.) B.S.P.) are genetically and morphologically similar but ecologically distinct species. We determined intraspecific seed-source and interspecific variation of red spruce and black spruce, from across the near-northern margins of their ranges, for several light-energy processing and freezing-tolerance adaptive traits. Before exposure to low temperature, red spruce had variable fluorescence (Fv) similar to black spruce, but higher photochemical efficiency (Fv/Fm), lower quantum yield, lower chlorophyll fluorescence (%), and higher thermal dissipation efficiency (qN), although the seed-source effect and the seed-source x species interaction were significant only for Fv/Fm. After low-temperature exposure (-40 degrees C), red spruce had significantly lower Fv/Fm, quantum yield and qN than black spruce, but higher chlorophyll fluorescence and relative fluorescence. Species, seed-source effect, and seed-source x species interaction were consistent with predictions based on genetic (e.g., geographic) origins. Multi-temperature exposures (5, -20 and -40 degrees C) often produced significant species and temperature effects, and species x temperature interactions as a result of species-specific responses to temperature exposures. The inherent physiological species-specific adaptations of red spruce and black spruce were largely consistent with a shade-tolerant, late-successional species and an early successional species, respectively. Species differences in physiological adaptations conform to a biological trade-off, probably as a result of natural selection pressure in response to light availability and prevailing temperature gradients.

Red fluorescence of dental plaque in children -A cross-sectional study.

PubMed

Volgenant, Catherine M C; Zaura, Egija; Brandt, Bernd W; Buijs, Mark J; Tellez, Marisol; Malik, Gayatri; Ismail, Amid I; Ten Cate, Jacob M; van der Veen, Monique H

2017-03-01

The relation between the presence of red fluorescent plaque and the caries status in children was studied. In addition, the microbial composition of dental plaque from sites with red fluorescent plaque (RFP) and from sites with no red fluorescent plaque (NFP) was assessed. Fluorescence photographs were taken from fifty children (6-14 years old) with overnight plaque. Full-mouth caries scores (ICDAS II) were obtained. The composition of a saliva sample and two plaque samples (RFP and NFP) was assessed using 16S rDNA sequencing. At the site level, no clinically relevant correlations were found between the presence of RFP and the caries status. At the subject level, a weak correlation was found between RFP and the caries status when non-cavitated lesions were included (r s =0.37, p=0.007). The microbial composition of RFP differed significantly from NFP. RFP had more anaerobes and more Gram-negative bacterial taxa. The most discriminative operational taxonomic units (OTUs) for RFP were Corynebacterium, Leptotrichia, Porphyromonas and Selenomonas, while the most discriminative OTUs for NFP were Neisseria, Actinomyces, Streptococcus and Rothia. There were no clinical relevant correlations in this cross-sectional study between the presence of RFP and (early) caries lesions. There were differences in the composition of these phenotypically different plaque samples: RFP contained more Gram-negative, anaerobic taxa and was more diverse than NFP. The study outcomes provide more insight in the possibilities to use plaque fluorescence in oral health risk assessments. Copyright © 2017 Elsevier Ltd. All rights reserved.

Pigment characterization of important golden age panel paintings of the 17th century

NASA Astrophysics Data System (ADS)

Pięta, Ewa; Proniewicz, Edyta; Szmelter-Fausek, Bożena; Olszewska-Świetlik, Justyna; Proniewicz, Leonard M.

2015-02-01

Samples were obtained from two world-famous 17th century panel paintings of the Gdańsk school of panting: 'Seven Acts of Charity' (1607, in St. Mary's Church in Gdańsk, Poland) by Anton Möller and 'Angelic Concert' (1611, in Diocesan Museum in Pelplin, Poland) by Hermann Han. Micro-Raman spectroscopy (MRS), optical microscopy (OM), and X-ray fluorescence (XRF) spectroscopy studies of the samples were performed to characterize the pigments present in the individual painting layers (a rich palette of white, black, blue, red, and yellow pigments) and the pictorial techniques used by the artists.

Bright and photostable cyanine-styryl chromophores with green and red fluorescence colour for DNA staining

NASA Astrophysics Data System (ADS)

Bohländer, Peggy R.; Wagenknecht, Hans-Achim

2015-12-01

The synthesis and optical characterisation of a series of green- and red-emitting cyanine and cyanine-styryl dyes is presented that were developed based on the cyanine-indole-quinolinium and based on the thiazole red type structure. For the green emitting fluorophores the quinolinium part was replaced by a pyridinium group. The bridge to the indole group was attached either to the 2-position or to the 4-position of the pyridinium moiety. For the red-emitting dyes the connection to the indole moiety is at the 4-position of the quinolinium part. In each set of dyes a methyl group at the indole-NH and/or a phenyl group at the 2-position of the indole part were introduced to tune the optical properties and photostability. Additionally, two dyes were modified with a cyano group to tune the photophysical properties and to enhance the photostabilities. The developed dyes show good photostabilities and bright green or red fluorescence intensities in the presence of DNA. Thus, these dyes represent important and promising candidates for fluorescent molecular imaging of nucleic acids inside living cells.

Fluorescence from Multiple Chromophore Hydrogen-Bonding States in the Far-Red Protein TagRFP675.

PubMed

Konold, Patrick E; Yoon, Eunjin; Lee, Junghwa; Allen, Samantha L; Chapagain, Prem P; Gerstman, Bernard S; Regmi, Chola K; Piatkevich, Kiryl D; Verkhusha, Vladislav V; Joo, Taiha; Jimenez, Ralph

2016-08-04

Far-red fluorescent proteins are critical for in vivo imaging applications, but the relative importance of structure versus dynamics in generating large Stokes-shifted emission is unclear. The unusually red-shifted emission of TagRFP675, a derivative of mKate, has been attributed to the multiple hydrogen bonds with the chromophore N-acylimine carbonyl. We characterized TagRFP675 and point mutants designed to perturb these hydrogen bonds with spectrally resolved transient grating and time-resolved fluorescence (TRF) spectroscopies supported by molecular dynamics simulations. TRF results for TagRFP675 and the mKate/M41Q variant show picosecond time scale red-shifts followed by nanosecond time blue-shifts. Global analysis of the TRF spectra reveals spectrally distinct emitting states that do not interconvert during the S1 lifetime. These dynamics originate from photoexcitation of a mixed ground-state population of acylimine hydrogen bond conformers. Strategically tuning the chromophore environment in TagRFP675 might stabilize the most red-shifted conformation and result in a variant with a larger Stokes shift.

Synthesizing a nano-composite of BSA-capped Au nanoclusters/graphitic carbon nitride nanosheets as a new fluorescent probe for dopamine detection.

PubMed

Guo, Xinrong; Wu, Fangying; Ni, Yongnian; Kokot, Serge

2016-10-26

A strong red fluorescent nanocomposite, consisting of graphite-like carbon nitride nanosheets (g-C 3 N 4 NSs) and serum albumin-capped Au nanoclusters (AuNCs), was synthesized. Dopamine (DA) can quench the red fluorescence of the nanocomposite, based on the Forster resonance energy transfer (FRET) mechanism. In this quenching process, the energy is transferred from the fluorescent g-C 3 N 4 NSs-AuNCs to the oxidized DA quinine molecules (DA is easily oxidated to form DA quinine in air). The red fluorescence emission at 420 nm decreases dramatically and the quenching ratio (F 0 - F)/F 0 is linearly related to the concentration of DA in the range of 0.05-8.0 μmol L -1 with a detection limit of 0.018 μmol L -1  (S/N = 3). Additionally, this sensor has a potential of application to assay the DA in the real samples, such as human serum and human urine. Copyright © 2016 Elsevier B.V. All rights reserved.

Theoretical Design of a Two-Photon Fluorescent Probe for Nitric Oxide with Enhanced Emission Induced by Photoninduced Electron Transfer.

PubMed

Zhang, Yujin; Leng, Jiancai; Hu, Wei

2018-04-25

In the present work, we systematically investigate the sensing abilities of two recently literature-reported two-photon fluorescent NO probes, i.e., the o-phenylenediamine derivative of Nile Red and the p-phenylenediamine derivative of coumarin. The recognition mechanisms of these probes are studied by using the molecular orbital classifying method, which demonstrates the photoinduced electron transfer process. In addition, we have designed two new probes by swapping receptor units present on fluorophores, i.e., the p-phenylenediamine derivative of Nile Red and the o-phenylenediamine derivative of coumarin. However, it illustrates that only the latter has ability to function as off-on typed fluorescent probe for NO. More importantly, calculations on the two-photon absorption properties of the probes demonstrate that both receptor derivatives of coumarin possess larger TPA cross-sections than Nile Red derivatives, which makes a better two photon fluorescent probe. Our theoretical investigations reveal that the underlying mechanism satisfactorily explain the experimental results, providing a theoretical basis on the structure-property relationships which is beneficial to developing new two-photon fluorescent probes for NO.

Fluorescent Proteins as Biomarkers and Biosensors: Throwing Color Lights on Molecular and Cellular Processes

PubMed Central

Stepanenko, Olesya V.; Verkhusha, Vladislav V.; Kuznetsova, Irina M.; Uversky, Vladimir N.; Turoverov, K.K.

2010-01-01

Green fluorescent protein (GFP) from jellyfish Aequorea victoria is the most extensively studied and widely used in cell biology protein. GFP-like proteins constitute a fast growing family as several naturally occurring GFP-like proteins have been discovered and enhanced mutants of Aequorea GFP have been created. These mutants differ from wild-type GFP by conformational stability, quantum yield, spectroscopic properties (positions of absorption and fluorescence spectra) and by photochemical properties. GFP-like proteins are very diverse, as they can be not only green, but also blue, orange-red, far-red, cyan, and yellow. They also can have dual-color fluorescence (e.g., green and red) or be non-fluorescent. Some of them possess kindling property, some are photoactivatable, and some are photoswitchable. This review is an attempt to characterize the main color groups of GFP-like proteins, describe their structure and mechanisms of chromophore formation, systemize data on their conformational stability and summarize the main trends of their utilization as markers and biosensors in cell and molecular biology. PMID:18691124

Optical spectroscopy for stereotactic biopsy of brain tumors

NASA Astrophysics Data System (ADS)

Markwardt, Niklas; von Berg, Anna; Fiedler, Sebastian; Goetz, Marcus; Haj-Hosseini, Neda; Polzer, Christoph; Stepp, Herbert; Zelenkov, Petr; Rühm, Adrian

2015-07-01

Stereotactic biopsy procedure is performed to obtain a tissue sample for diagnosis purposes. Currently, a fiber-based mechano-optical device for stereotactic biopsies of brain tumors is developed. Two different fluorophores are employed to improve the safety and reliability of this procedure: The fluorescence of intravenously applied indocyanine green (ICG) facilitates the recognition of blood vessels and thus helps minimize the risk of cerebral hemorrhages. 5- aminolevulinic-acid-induced protoporphyrin IX (PpIX) fluorescence is used to localize vital tumor tissue. ICG fluorescence detection using a 2-fiber probe turned out to be an applicable method to recognize blood vessels about 1.5 mm ahead of the fiber tip during a brain tumor biopsy. Moreover, the suitability of two different PpIX excitation wavelengths regarding practical aspects was investigated: While PpIX excitation in the violet region (at 405 nm) allows for higher sensitivity, red excitation (at 633 nm) is noticeably superior with regard to blood layers obscuring the fluorescence signal. Contact measurements on brain simulating agar phantoms demonstrated that a typical blood coverage of the tumor reduces the PpIX signal to about 75% and nearly 0% for 633 nm and 405 nm excitation, respectively. As a result, 633 nm seems to be the wavelength of choice for PpIX-assisted detection of high-grade gliomas in stereotactic biopsy.

Red Fluorescent Protein-Aequorin Fusions as Improved Bioluminescent Ca2+ Reporters in Single Cells and Mice

PubMed Central

Bakayan, Adil; Vaquero, Cecilia F.; Picazo, Fernando; Llopis, Juan

2011-01-01

Bioluminescence recording of Ca2+ signals with the photoprotein aequorin does not require radiative energy input and can be measured with a low background and good temporal resolution. Shifting aequorin emission to longer wavelengths occurs naturally in the jellyfish Aequorea victoria by bioluminescence resonance energy transfer (BRET) to the green fluorescent protein (GFP). This process has been reproduced in the molecular fusions GFP-aequorin and monomeric red fluorescent protein (mRFP)-aequorin, but the latter showed limited transfer efficiency. Fusions with strong red emission would facilitate the simultaneous imaging of Ca2+ in various cell compartments. In addition, they would also serve to monitor Ca2+ in living organisms since red light is able to cross animal tissues with less scattering. In this study, aequorin was fused to orange and various red fluorescent proteins to identify the best acceptor in red emission bands. Tandem-dimer Tomato-aequorin (tdTA) showed the highest BRET efficiency (largest energy transfer critical distance R0) and percentage of counts in the red band of all the fusions studied. In addition, red fluorophore maturation of tdTA within cells was faster than that of other fusions. Light output was sufficient to image ATP-induced Ca2+ oscillations in single HeLa cells expressing tdTA. Ca2+ rises caused by depolarization of mouse neuronal cells in primary culture were also recorded, and changes in fine neuronal projections were spatially resolved. Finally, it was also possible to visualize the Ca2+ activity of HeLa cells injected subcutaneously into mice, and Ca2+ signals after depositing recombinant tdTA in muscle or the peritoneal cavity. Here we report that tdTA is the brightest red bioluminescent Ca2+ sensor reported to date and is, therefore, a promising probe to study Ca2+ dynamics in whole organisms or tissues expressing the transgene. PMID:21589654

Structure of the red fluorescent protein from a lancelet (Branchiostoma lanceolatum): a novel GYG chromophore covalently bound to a nearby tyrosine

DOE Office of Scientific and Technical Information (OSTI.GOV)

Pletnev, Vladimir Z., E-mail: vzpletnev@gmail.com; Pletneva, Nadya V.; Lukyanov, Konstantin A.

The crystal structure of the novel red emitting fluorescent protein from lancelet Branchiostoma lanceolatum (Chordata) revealed an unusual five residues cyclic unit comprising Gly58-Tyr59-Gly60 chromophore, the following Phe61 and Tyr62 covalently bound to chromophore Tyr59. A key property of proteins of the green fluorescent protein (GFP) family is their ability to form a chromophore group by post-translational modifications of internal amino acids, e.g. Ser65-Tyr66-Gly67 in GFP from the jellyfish Aequorea victoria (Cnidaria). Numerous structural studies have demonstrated that the green GFP-like chromophore represents the ‘core’ structure, which can be extended in red-shifted proteins owing to modifications of the protein backbonemore » at the first chromophore-forming position. Here, the three-dimensional structures of green laGFP (λ{sub ex}/λ{sub em} = 502/511 nm) and red laRFP (λ{sub ex}/λ{sub em} ≃ 521/592 nm), which are fluorescent proteins (FPs) from the lancelet Branchiostoma lanceolatum (Chordata), were determined together with the structure of a red variant laRFP-ΔS83 (deletion of Ser83) with improved folding. Lancelet FPs are evolutionarily distant and share only ∼20% sequence identity with cnidarian FPs, which have been extensively characterized and widely used as genetically encoded probes. The structure of red-emitting laRFP revealed three exceptional features that have not been observed in wild-type fluorescent proteins from Cnidaria reported to date: (i) an unusual chromophore-forming sequence Gly58-Tyr59-Gly60, (ii) the presence of Gln211 at the position of the conserved catalytic Glu (Glu222 in Aequorea GFP), which proved to be crucial for chromophore formation, and (iii) the absence of modifications typical of known red chromophores and the presence of an extremely unusual covalent bond between the Tyr59 C{sup β} atom and the hydroxyl of the proximal Tyr62. The impact of this covalent bond on the red emission and the large Stokes shift (∼70 nm) of laRFP was verified by extensive structure-based site-directed mutagenesis.« less

Formation of hemoglobin photoproduct is responsible for two-photon and single photon-excited fluorescence of red blood cells

NASA Astrophysics Data System (ADS)

Shirshin, Evgeny A.; Yakimov, Boris P.; Rodionov, Sergey A.; Omelyanenko, Nikolai P.; Priezzhev, Alexander V.; Fadeev, Victor V.; Lademann, Juergen; Darvin, Maxim E.

2018-07-01

Two-photon excited fluorescence of red blood cells (RBC) has been reported to be applicable for their assessment in vitro and in vivo. The corresponding fluorescence emission was ascribed to hemoglobin (Hb), however, as Hb is essentially non-fluorescent at single-photon excitation, the mechanism of two-photon excited fluorescence of RBC remains debatable. Here we show that a fluorescent photoproduct, characterized by an ultrafast decay of excitation, is formed after irradiation of Hb with femtosecond laser pulses with ca. 8 · 10‑5 quantum yield, and that it is also fluorescent at single-photon excitation. The formation of a similar photoproduct was also shown for Hb continuous wave irradiation with blue light with ca. 10‑5 formation quantum yield. The kinetics of the Hb photoproduct formation and its spectral properties were investigated. The obtained results clarify the processes responsible for RBC fluorescence observed in two-photon microscopy experiments.

Theoretical investigation for excitation light and fluorescence signal of fiber optical sensor using tapered fiber tip.

PubMed

Yuan, Yinquan; Ding, Liyun

2011-10-24

For fiber optical sensor made of tapered fiber tip, the effects of the geometrical parameters of tapered tip on two important factors have been investigated. One factor is the intensity of the evanescent wave into fluorescent layer through core-medium interface; the other is the intensity of fluorescence signal transmitted from fluorescent layer to measurement end. A dependence relation of the intensity of fluorescence signal transmitted from fluorescent layer to measurement end upon the geometrical parameters of tapered tip has been obtained. Theoretical results show that the intensity of the evanescent wave into fluorescent layer rises with the decrease of the end diameter of tapered tip, and the increase of the tip length; and the transmitted power of fluorescence signal increases linearly with the increase of the tip length due to the contribution of the side area of tapered tip. © 2011 Optical Society of America

Tracking the Invasion of Small Numbers of Cells in Paper-Based Assays with Quantitative PCR.

PubMed

Truong, Andrew S; Lochbaum, Christian A; Boyce, Matthew W; Lockett, Matthew R

2015-11-17

Paper-based scaffolds are an attractive material for culturing mammalian cells in a three-dimensional environment. There are a number of previously published studies, which utilize these scaffolds to generate models of aortic valves, cardiac ischemia and reperfusion, and solid tumors. These models have largely relied on fluorescence imaging and microscopy to quantify cells in the scaffolds. We present here a polymerase chain reaction (PCR)-based method, capable of quantifying multiple cell types in a single culture with the aid of DNA barcodes: unique sequences of DNA introduced to the genome of individual cells or cell types through lentiviral transduction. PCR-based methods are highly specific and are amenable to high-throughput and multiplexed analyses. To validate this method, we engineered two different breast cancer lines to constitutively express either a green or red fluorescent protein. These cells lines allowed us to directly compare the ability of fluorescence imaging (of the fluorescent proteins) and qPCR (of the unique DNA sequences of the fluorescent proteins) to quantify known numbers of cells in the paper based-scaffolds. We also used both methods to quantify the distribution of these breast cell lines in homotypic and heterotypic invasion assays. In the paper-based invasion assays, a single sheet of paper containing cells suspended in a hydrogel was sandwiched between sheets of paper containing only hydrogel. The stack was incubated, and the cells invaded the adjacent layers. The individual sheets of the invasion assay were then destacked and the number of cells in each layer quantified. Our results show both methods can accurately detect cell populations of greater than 500 cells. The qPCR method can repeatedly and accurately detect as few as 50 cells, allowing small populations of highly invasive cells to be detected and differentiated from other cell types.

Treatment of TNT red water by layer melt crystallization.

PubMed

Jo, Jeong-Hyeon; Ernest, Takyi; Kim, Kwang-Joo

2014-09-15

Treatment of the red water, which is wastewater of 2,4,6- trinitrotoluene (TNT) manufacturing process has been explored using ice crystallization. This study focuses on the formation of ice crystals from the red water in a layer crystallizer under various operating conditions. Among the parameters which affect layer crystallization, attention was given to cooling rate, cooling temperature, sweating rate and concentration of the red water. The study highlights the effect of subcooling and growth rate on purity of the ice crystalline layers produced. After sweating, the COD value of crystalline ice layer was significantly reduced from 10,000 mg/L to below 20mg/L. Most organic contaminants were removed in sweating fractions of 0.5. Eventually, the red water was treated by layer crystallization combined with the sweating process. Copyright © 2014 Elsevier B.V. All rights reserved.

Establishing and validating the fluorescent amyloid ligand h-FTAA (heptamer formyl thiophene acetic acid) to identify transthyretin amyloid deposits in carpal tunnel syndrome.

PubMed

Hahn, Katharina; Nilsson, K Peter R; Hammarström, Per; Urban, Peter; Meliss, Rolf Rüdiger; Behrens, Hans-Michael; Krüger, Sandra; Röcken, Christoph

2017-06-01

Transthyretin-derived (ATTR) amyloidosis is a frequent finding in carpal tunnel syndrome. We tested the following hypotheses: the novel fluorescent amyloid ligand heptameric formic thiophene acetic acid (h-FTAA) has a superior sensitivity for the detection of amyloid compared with Congo red-staining; Amyloid load correlates with patient gender and/or patient age. We retrieved 208 resection specimens obtained from 184 patients with ATTR amyloid in the carpal tunnel. Serial sections were stained with Congo red, h-FTAA and an antibody directed against transthyretin (TTR). Stained sections were digitalized and forwarded to computational analyses. The amount of amyloid was correlated with patient demographics. Amyloid stained intensely with h-FTAA and an anti-TTR-antibody. Congo red-staining combined with fluorescence microscopy was significantly less sensitive than h-FTAA-fluorescence and TTR-immunostaining: the highest percentage area was found in TTR-immunostained sections, followed by h-FTAA and Congo red. The Pearson correlation coefficient was .8 (Congo red vs. h-FTAA) and .9 (TTR vs. h-FTAA). Amyloid load correlated with patient gender, anatomical site and patient age. h-FTAA is a highly sensitive method to detect even small amounts of ATTR amyloid in the carpal tunnel. The staining protocol is easy and h-FTAA may be a much more sensitive procedure to detect amyloid at an earlier stage.

Retracing Evolution of Red Fluorescence in GFP-Like Proteins from Faviina Corals

PubMed Central

Field, Steven F.; Matz, Mikhail V.

2010-01-01

Proteins of the green fluorescent protein family represent a convenient experimental model to study evolution of novelty at the molecular level. Here, we focus on the origin of Kaede-like red fluorescent proteins characteristic of the corals of the Faviina suborder. We demonstrate, using an original approach involving resurrection and analysis of the library of possible evolutionary intermediates, that it takes on the order of 12 mutations, some of which strongly interact epistatically, to fully recapitulate the evolution of a red fluorescent phenotype from the ancestral green. Five of the identified mutations would not have been found without the help of ancestral reconstruction, because the corresponding site states are shared between extant red and green proteins due to their recent descent from a dual-function common ancestor. Seven of the 12 mutations affect residues that are not in close contact with the chromophore and thus must exert their effect indirectly through adjustments of the overall protein fold; the relevance of these mutations could not have been anticipated from the purely theoretical analysis of the protein's structure. Our results introduce a powerful experimental approach for comparative analysis of functional specificity in protein families even in the cases of pronounced epistasis, provide foundation for the detailed studies of evolutionary trajectories leading to novelty and complexity, and will help rational modification of existing fluorescent labels. PMID:19793832

Highly efficient red OLEDs using DCJTB as the dopant and delayed fluorescent exciplex as the host.

PubMed

Zhao, Bo; Zhang, Tianyou; Chu, Bei; Li, Wenlian; Su, Zisheng; Wu, Hairuo; Yan, Xingwu; Jin, Fangming; Gao, Yuan; Liu, Chengyuan

2015-05-29

In this manuscript, we demonstrated a highly efficient DCJTB emission with delayed fluorescent exciplex TCTA:3P-T2T as the host. For the 1.0% DCJTB doped concentration, a maximum luminance, current efficiency, power efficiency and EQE of 22,767 cd m(-2), 22.7 cd A(-1), 21.5 lm W(-1) and 10.15% were achieved, respectively. The device performance is the best compared to either red OLEDs with traditional fluorescent emitter or traditional red phosphor of Ir(piq)3 doped into CBP host. The extraction of so high efficiency can be explained as the efficient triplet excitons up-conversion of TCTA:3P-T2T and the energy transfer from exciplex host singlet state to DCJTB singlet state.

Canopy Level Solar Induced Fluorescence for Vegetation in Controlled Experiments

NASA Technical Reports Server (NTRS)

Middleton, E. M.; Corp, L. A.; Campbell, P. K. Entcheva

2007-01-01

Solar induced chlorophyll fluorescence (SIF) was retrieved from high resolution reflectance spectra acquired one meter above saplings of three deciduous tree species during springtime (three weeks after leaf flush) and in late summer when foliage was mature. SIF was determined by application of the Fraunhofer Line Depth (FLD) Principal to above-canopy spectra acquired with an Analytical Spectral Devices (ASD) Fieldspec spectroradiometer (3.2 nm resolution with 1.2 nm sampling interval). SIF retrievals were made at the two atmospheric oxygen (O2) absorption features that occur in the chlorophyll fluorescence (ChlF) region (660 -780 nm). These telluric features are 02V, the broader and deeper feature centered at 760 nm, but located on the shoulder of the far-red ChlF peak at 740 nm; and 023, a narrow feature centered at 688 nm that is positioned near the red ChlF peak at 685 nm. Supporting, coincident leaf level fluorescence, reflectance, photochemical and other measurements were also made. At the leaf level, these measurements included in situ photosynthetic capacity (Pmax) and light adapted total chlorophyll fluorescence (Fs') collected at steady state under high light and controlled chamber conditions (e.g., temperature, PAR, humidity, and COz); optical properties (reflectance, transmittance, absorptance); chlorophyll and carotenoid content; specific leaf mass; carbon (C) and nitrogen (N) content; fluorescence emission spectra at multiple excitation wavelengths; the ChlF contribution to red (R) and far-red (FR) reflectance; fluorescence imagery; and fluorescence excitation-emission matrices (EEMs). The tree species examined were tulip poplar (Liriodendron tulipifera L.), red maple (Acer rubrum L.), and sweetgum (Liquidambar styraczflua L.), and each had been provided four levels of N augmentation (0, 19, 37, and 75 kg Nhectare seasonally) to simulate atmospheric deposition from air pollution. Whole-plant SIF measurements of these species were compared with SIF estimates derived using FluorMOD, a radiative transfer model that includes fluorescence properties of foliage, when provided with our supporting measurements. Simulated values for SIF were also compared with similar estimates made over three years for corn (Zea mays L) crops under N treatments (20, 50,70, and 140 kg Nhectare) and with corn and pepper (Capsicum annuum L.) plants provided dimethyl urea (DCMU, 0 and 5 x 10" M) which were grown in pots and grouped in artificial canopies for spectral measurements. For near-surface measurements of healthy and stressed vegetation, red SIF retrieved at 688 nm (023) varied between 2-7 mW/m(exp 2)/nm/sr while far-red SIF retrieved at 760 nm (O2A) varied between 0.5 and 4.0 mW/m(exp 2)/nm/sr. Typical values for the SIF red/far-red ratio ranged between 1.75 and 4.0. Relationships of SIF to spectral reflectance indices and foliar photochemical indices were examined, such as photosynthetic light use efficiency. Initial investigations of the variability in these measurements at the same leaf temperature for sunlit and shaded foliage showed us that shaded foliage produced higher ChlF and light use efficiency. Other factors that affect SIF determinations are discussed. These results will assist in determine the expected intensity of the SIF signal from vegetation near the surface, as well as to identify enhancements needed for FluorMOD (or other such models), and will assist in determining relationships of SIF to reflectance indices and carbon dynamics.

Carbazole-based BODIPYs with ethynyl substituents at the boron center: solid-state excimer fluorescence in the VIS/NIR region.

PubMed

Maeda, Chihiro; Nagahata, Keiji; Ema, Tadashi

2017-09-26

Carbazole-based BODIPYs 1-6 with several different substituents at the boron atom site were synthesized. These dyes fluoresced in the solid state, and 3a with phenylethynyl groups exhibited a red-shifted and broad fluorescence spectrum, which suggested an excimer emission. Its derivatives 3b-n were synthesized, and the relationship between the solid-state emission and crystal packing was investigated. The X-ray crystal structures revealed cofacial dimers that might form excimers. From the structural optimization results, we found that the introduction of mesityl groups hindered intermolecular access and led to reduced interactions between the dimers. In addition, the red-shifted excimer fluorescence suppressed self-absorption, and dyes with ethynyl groups showed solid-state fluorescence in the vis/NIR region.

Molecular and structural characterization of New Red and Erythrosine by fluorescence polarization spectroscopy

NASA Astrophysics Data System (ADS)

Zhu, Chun; Du, Jia-Meng; Zhao, Jin-Chen; Zhu, Tuo; Chen, Guo-Qing

2017-07-01

The fundamental and the fluorescence anisotropies of New Red and Erythrosine were measured. The intersection angles between the absorption and the emission dipole moments for New Red and Erythrosine are 4.44∘ and 23.26∘, respectively. The average angle shift of the emission dipole moment of New Red is 3.91∘ during the lifetime of the excited state. This indicates that it has a bifurcated linear structure with weak rotational capacity. The average angle shift of the emission dipole moment of Erythrosine is 9.25∘, indicating that it has a partial planar structure and is easier to rotate. The spatial ground state structures were simulated with Gaussian 09.

Fluorescence detection and photodynamic activity of endogenous protoporphyrin in human skin

NASA Astrophysics Data System (ADS)

Koenig, Karsten; Rueck, Angelika C.; Schneckenburger, Herbert

1992-07-01

Human skin shows a strong autofluorescence in the red spectral region with main peaks around 600, 620, and 640 nm caused by the porphyrin production of the gram positive lipophile skin bacterium Propionibacterium acnes. Irradiation of these bacteria reduces the integral fluorescence intensity and induces the formation of photoproducts with fluorescence bands around 670 nm and decay times of about 1 and 5 ns. The photoproduct formation is connected with an increased absorption in the red spectral region. The endogenous fluorescent porphyrins act as photosensitizers. Photodestruction of Propionibacteria acnes by visible light appears therefore to be a promising therapy. The photodynamic activity of the photoproducts was lower than that of protoporphyrin IX.

The influence of surface plasmons on fluorescence of the dye Lumogen F red 300 in condensed phase

NASA Astrophysics Data System (ADS)

Reisfeld, Renata; Levchenko, Viktoria

2017-01-01

The paper is divided to two parts one is a short description of scientific activity of Georges Boulon and my long standing friendship with him, the second part consists of the new findings of increasing fluorescence by surface plasmons studies performed recently in Jerusalem. We describe the synthesis of copper nanoparticles and their incorporation into polyvinylpyrrolidone and into sol-gel matrix co-doped with the fluorescent dye Lumogen F Red 300. The absorption and emission spectra of samples undoped and co-doped with nanoparticles reveal the increase of fluorescence of the dye in presence of copper nanoparticles. The phenomenon arises from scattered light by the nanoparticles.

Red fluorescent genetically encoded indicator for intracellular hydrogen peroxide

NASA Astrophysics Data System (ADS)

Ermakova, Yulia G.; Bilan, Dmitry S.; Matlashov, Mikhail E.; Mishina, Natalia M.; Markvicheva, Ksenia N.; Subach, Oksana M.; Subach, Fedor V.; Bogeski, Ivan; Hoth, Markus; Enikolopov, Grigori; Belousov, Vsevolod V.

2014-10-01

Reactive oxygen species (ROS) are conserved regulators of numerous cellular functions, and overproduction of ROS is a hallmark of various pathological processes. Genetically encoded fluorescent probes are unique tools to study ROS production in living systems of different scale and complexity. However, the currently available recombinant redox sensors have green emission, which overlaps with the spectra of many other probes. Expanding the spectral range of recombinant in vivo ROS probes would enable multiparametric in vivo ROS detection. Here we present the first genetically encoded red fluorescent sensor for hydrogen peroxide detection, HyPerRed. The performance of this sensor is similar to its green analogues. We demonstrate the utility of the sensor by tracing low concentrations of H2O2 produced in the cytoplasm of cultured cells upon growth factor stimulation. Moreover, using HyPerRed we detect local and transient H2O2 production in the mitochondrial matrix upon inhibition of the endoplasmic reticulum Ca2+ uptake.

A set of GFP-based organelle marker lines combined with DsRed-based gateway vectors for subcellular localization study in rice (Oryza sativa L.).

PubMed

Wu, Tsung-Meng; Lin, Ke-Chun; Liau, Wei-Shiang; Chao, Yun-Yang; Yang, Ling-Hung; Chen, Szu-Yun; Lu, Chung-An; Hong, Chwan-Yang

2016-01-01

In the post-genomic era, many useful tools have been developed to accelerate the investigation of gene functions. Fluorescent proteins have been widely used as protein tags for studying the subcellular localization of proteins in plants. Several fluorescent organelle marker lines have been generated in dicot plants; however, useful and reliable fluorescent organelle marker lines are lacking in the monocot model rice. Here, we developed eight different GFP-based organelle markers in transgenic rice and created a set of DsRed-based gateway vectors for combining with the marker lines. Two mitochondrial-localized rice ascorbate peroxidase genes fused to DsRed and successfully co-localized with mitochondrial-targeted marker lines verified the practical use of this system. The co-localization of GFP-fusion marker lines and DsRed-fusion proteins provide a convenient platform for in vivo or in vitro analysis of subcellular localization of rice proteins.

Non-invasive intravital imaging of cellular differentiation with a bright red-excitable fluorescent protein

PubMed Central

Chu, Jun; Haynes, Russell D; Corbel, Stéphane Y; Li, Pengpeng; González-González, Emilio; Burg, John S; Ataie, Niloufar J; Lam, Amy J; Cranfill, Paula J; Baird, Michelle A; Davidson, Michael W; Ng, Ho-Leung; Garcia, K Christopher; Contag, Christopher H; Shen, Kang; Blau, Helen M; Lin, Michael Z

2014-01-01

A method for non-invasive visualization of genetically labelled cells in animal disease models with micron-level resolution would greatly facilitate development of cell-based therapies. Imaging of fluorescent proteins (FPs) using red excitation light in the “optical window” above 600 nm is one potential method for visualizing implanted cells. However, previous efforts to engineer FPs with peak excitation beyond 600 nm have resulted in undesirable reductions in brightness. Here we report three new red-excitable monomeric FPs obtained by structure-guided mutagenesis of mNeptune, previously the brightest monomeric FP when excited beyond 600 nm. Two of these, mNeptune2 and mNeptune2.5, demonstrate improved maturation and brighter fluorescence, while the third, mCardinal, has a red-shifted excitation spectrum without reduction in brightness. We show that mCardinal can be used to non-invasively and longitudinally visualize the differentiation of myoblasts and stem cells into myocytes in living mice with high anatomical detail. PMID:24633408

Preparation of Au Nanoclusters-Modified Polylactic Acid Fiber with Bright Red Fluorescence and its Use as Sensing Probe.

PubMed

Zhu, Wenli; Li, Huili; Wan, Ajun; Liu, Lanbo

2017-01-01

In present work, the Au nanoclusters-modified polylactic acid fiber (PLA-Au NCs) with bright red fluorescence were fabricated by the encapsulation of Au nanoclusters (Au NCs) in the PLA fiber treated with H 2 O 2 . The Au 25 nanoclusters stabilized by bovine serum albumin (BSA-Au NCs) were prepared via an improved "green" synthetic routine. With pretreatment of the PLA fiber in H 2 O 2 concentration of 12 and 18 %, the as-prepared PLA-Au NCs exhibited brighter red emission with a strong peak centered at ~640 nm than BSA-Au NCs. The fluorescence can be quenched by nitric oxide (NO). A good linear relationship between the relative fluorescence quenching intensity of the as-prepared PLA-Au NCs and the concentration of NO can be obtained in the range of 0.0732 to 0.7320 mM, and the detection limit was 0.0070 mM.

Distinct Interfacial Fluorescence in Oil-in-Water Emulsions via Exciton Migration of Conjugated Polymers.

PubMed

Koo, Byungjin; Swager, Timothy M

2017-09-01

Commercial dyes are extensively utilized to stain specific phases for the visualization applications in emulsions and bioimaging. In general, dyes emit only one specific fluorescence signal and thus, in order to stain various phases and/or interfaces, one needs to incorporate multiple dyes and carefully consider their compatibility to avoid undesirable interactions with each other and with the components in the system. Herein, surfactant-type, perylene-endcapped fluorescent conjugated polymers that exhibit two different emissions are reported, which are cyan in water and red at oil-water interfaces. The interfacially distinct red emission results from enhanced exciton migration from the higher-bandgap polymer backbone to the lower-bandgap perylene endgroup. The confocal microscopy images exhibit the localized red emission exclusively from the circumference of oil droplets. This exciton migration and dual fluorescence of the polymers in different physical environments can provide a new concept of visualization methods in many amphiphilic colloidal systems and bioimaging. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Photoswitchable red fluorescent protein with a large Stokes shift

PubMed Central

Piatkevich, Kiryl D.; English, Brian P.; Malashkevich, Vladimir N.; Xiao, Hui; Almo, Steven C.; Singer, Robert H.; Verkhusha, Vladislav V.

2014-01-01

SUMMARY Subclass of fluorescent proteins, large Stokes shift fluorescent proteins, is characterized by their increased spread between the excitation and emission maxima. Here we report a photoswitchable variant of a red fluorescent protein with a large Stokes shift, PSLSSmKate, which initially exhibits excitation/emission at 445/622 nm, but irradiation with violet light photoswitches PSLSSmKate into a common red form with excitation/emission at 573/621 nm. We characterize spectral, photophysical and biochemical properties of PSLSSmKate in vitro and in mammalian cells, and determine its crystal structure in the large Stokes shift form. Mass-spectrometry, mutagenesis and spectroscopic analysis of PSLSSmKate allow us to propose molecular mechanisms for the large Stokes shift, pH dependence and light-induced chromophore transformation. We demonstrate applicability of PSLSSmKate to superresolution PALM microscopy and protein dynamics in live cells. Given its promising properties, we expect that PSLSSmKate-like phenotype will be further used for photoactivatable imaging and tracking multiple populations of intracellular objects. PMID:25242289

Synthesis and characterization of colloidal fluorescent silver nanoclusters.

PubMed

Huang, Sherry; Pfeiffer, Christian; Hollmann, Jana; Friede, Sebastian; Chen, Justin Jin-Ching; Beyer, Andreas; Haas, Benedikt; Volz, Kerstin; Heimbrodt, Wolfram; Montenegro Martos, Jose Maria; Chang, Walter; Parak, Wolfgang J

2012-06-19

Ultrasmall water-soluble silver nanoclusters are synthesized, and their properties are investigated. The silver nanoclusters have high colloidal stability and show fluorescence in the red. This demonstrates that like gold nanoclusters also silver nanoclusters can be fluorescent.

Autofluorescence study and selected cyanidin quantification in the Jewel orchids Anoectochilus sp. and Ludisia discolor.

PubMed

Poobathy, Ranjetta; Zakaria, Rahmad; Murugaiyah, Vikneswaran; Subramaniam, Sreeramanan

2018-01-01

Anoectochilus sp. and Ludisia discolor are known as Jewel orchids. Both species are terrestrial wild orchids that grow in shaded areas of forests. The Jewel orchids are renowned for the beauty of their leaves, which are dark-green laced with silvery or golden veins. The orchids are used as a cure in various parts of Asia. Overharvesting and anthropogenic disturbances threaten the existence of the Jewel orchids in the wild, necessitating human intervention in their survival. An understanding of the structure and adaptations of a plant may assist in its survival when propagated outside of its habitat. In this study, ex vitro leaves of Anoectochilus sp. and L. discolor were subjected to freehand sectioning, and then inspected through brightfield and fluorescence microscopy. The study indicated that all parts of both plants presented typical monocotyledonous characteristics except the leaves. The leaves displayed dorsiventrality with distinct palisade and spongy mesophyll layers. The spongy mesophyll layer contained cells which fluoresced a bright red when exposed to ultraviolet, blue, and green light wavelengths, hinting at the presence of anthocyanins for photoprotection. Cyanidin was detected in the leaves of L. discolor, as enumerated through high performance liquid chromatography (HPLC). The observations indicated that Anoectochilus sp. and L. discolor are well-adapted to live under shaded conditions with minimal exposure to light.

Incomplete proteasomal degradation of green fluorescent proteins in the context of tandem fluorescent protein timers

PubMed Central

Khmelinskii, Anton; Meurer, Matthias; Ho, Chi-Ting; Besenbeck, Birgit; Füller, Julia; Lemberg, Marius K.; Bukau, Bernd; Mogk, Axel; Knop, Michael

2016-01-01

Tandem fluorescent protein timers (tFTs) report on protein age through time-dependent change in color, which can be exploited to study protein turnover and trafficking. Each tFT, composed of two fluorescent proteins (FPs) that differ in maturation kinetics, is suited to follow protein dynamics within a specific time range determined by the maturation rates of both FPs. So far, tFTs have been constructed by combining slower-maturing red fluorescent proteins (redFPs) with the faster-maturing superfolder green fluorescent protein (sfGFP). Toward a comprehensive characterization of tFTs, we compare here tFTs composed of different faster-maturing green fluorescent proteins (greenFPs) while keeping the slower-maturing redFP constant (mCherry). Our results indicate that the greenFP maturation kinetics influences the time range of a tFT. Moreover, we observe that commonly used greenFPs can partially withstand proteasomal degradation due to the stability of the FP fold, which results in accumulation of tFT fragments in the cell. Depending on the order of FPs in the timer, incomplete proteasomal degradation either shifts the time range of the tFT toward slower time scales or precludes its use for measurements of protein turnover. We identify greenFPs that are efficiently degraded by the proteasome and provide simple guidelines for the design of new tFTs. PMID:26609072

Silole-Based Red Fluorescent Organic Dots for Bright Two-Photon Fluorescence In vitro Cell and In vivo Blood Vessel Imaging.

PubMed

Chen, Bin; Feng, Guangxue; He, Bairong; Goh, Chiching; Xu, Shidang; Ramos-Ortiz, Gabriel; Aparicio-Ixta, Laura; Zhou, Jian; Ng, Laiguan; Zhao, Zujin; Liu, Bin; Tang, Ben Zhong

2016-02-10

Robust luminescent dyes with efficient two-photon fluorescence are highly desirable for biological imaging applications, but those suitable for organic dots fabrication are still rare because of aggregation-caused quenching. In this work, a red fluorescent silole, 2,5-bis[5-(dimesitylboranyl)thiophen-2-yl]-1-methyl-1,3,4-triphenylsilole ((MesB)2 DTTPS), is synthesized and characterized. (MesB)2 DTTPS exhibits enhanced fluorescence efficiency in nanoaggregates, indicative of aggregation-enhanced emission (AEE). The organic dots fabricated by encapsulating (MesB)2 DTTPS within lipid-PEG show red fluorescence peaking at 598 nm and a high fluorescence quantum yield of 32%. Upon excitation at 820 nm, the dots show a large two-photon absorption cross section of 3.43 × 10(5) GM, which yields a two-photon action cross section of 1.09 × 10(5) GM. These (MesB)2 DTTPS dots show good biocompatibility and are successfully applied to one-photon and two-photon fluorescence imaging of MCF-7 cells and two-photon in vivo visualization of the blood vascular of mouse muscle in a high-contrast and noninvasive manner. Moreover, the 3D blood vasculature located at the mouse ear skin with a depth of over 100 μm can also be visualized clearly, providing the spatiotemporal information about the whole blood vascular network. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Color stable white phosphorescent organic light emitting diodes with red emissive electron transport layer

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wook Kim, Jin; Yoo, Seung Il; Sung Kang, Jin

2015-06-28

We analyzed the performance of multi-emissive white phosphorescent organic light-emitting diodes (PHOLEDs) in relation to various red emitting sites of hole and electron transport layers (HTL and ETL). The shift of the recombination zone producing stable white emission in PHOLEDs was utilized as luminance was increased with red emission in its electron transport layer. Multi-emissive white PHOLEDs including the red light emitting electron transport layer yielded maximum external quantum efficiency of 17.4% with CIE color coordinates (−0.030, +0.001) shifting only from 1000 to 10 000 cd/m{sup 2}. Additionally, we observed a reduction of energy loss in the white PHOLED via Ir(piq){submore » 3} as phosphorescent red dopant in electron transport layer.« less

New methods for the retrieval of chlorophyll red fluorescence from hyperspectral satellite instruments: simulations and application to GOME-2 and SCIAMACHY

NASA Astrophysics Data System (ADS)

Joiner, Joanna; Yoshida, Yasuko; Guanter, Luis; Middleton, Elizabeth M.

2016-08-01

Global satellite measurements of solar-induced fluorescence (SIF) from chlorophyll over land and ocean have proven useful for a number of different applications related to physiology, phenology, and productivity of plants and phytoplankton. Terrestrial chlorophyll fluorescence is emitted throughout the red and far-red spectrum, producing two broad peaks near 683 and 736 nm. From ocean surfaces, phytoplankton fluorescence emissions are entirely from the red region (683 nm peak). Studies using satellite-derived SIF over land have focused almost exclusively on measurements in the far red (wavelengths > 712 nm), since those are the most easily obtained with existing instrumentation. Here, we examine new ways to use existing hyperspectral satellite data sets to retrieve red SIF (wavelengths < 712 nm) over both land and ocean. Red SIF is thought to provide complementary information to that from the far red for terrestrial vegetation. The satellite instruments that we use were designed to make atmospheric trace-gas measurements and are therefore not optimal for observing SIF; they have coarse spatial resolution and only moderate spectral resolution (0.5 nm). Nevertheless, these instruments, the Global Ozone Monitoring Instrument 2 (GOME-2) and the SCanning Imaging Absorption spectroMeter for Atmospheric CHartographY (SCIAMACHY), offer a unique opportunity to compare red and far-red terrestrial SIF at regional spatial scales. Terrestrial SIF has been estimated with ground-, aircraft-, or satellite-based instruments by measuring the filling-in of atmospheric and/or solar absorption spectral features by SIF. Our approach makes use of the oxygen (O2) γ band that is not affected by SIF. The SIF-free O2 γ band helps to estimate absorption within the spectrally variable O2 B band, which is filled in by red SIF. SIF also fills in the spectrally stable solar Fraunhofer lines (SFLs) at wavelengths both inside and just outside the O2 B band, which further helps to estimate red SIF emission. Our approach is then an extension of previous approaches applied to satellite data that utilized only the filling-in of SFLs by red SIF. We conducted retrievals of red SIF using an extensive database of simulated radiances covering a wide range of conditions. Our new algorithm produces good agreement between the simulated truth and retrievals and shows the potential of the O2 bands for noise reduction in red SIF retrievals as compared with approaches that rely solely on SFL filling. Biases seen with existing satellite data, most likely due to instrumental artifacts that vary in time, space, and with instrument, must be addressed in order to obtain reasonable results. Our 8-year record of red SIF observations over land with the GOME-2 allows for the first time reliable global mapping of monthly anomalies. These anomalies are shown to have similar spatiotemporal structure as those in the far red, particularly for drought-prone regions. There is a somewhat larger percentage response in the red as compared with the far red for these areas that are drought sensitive. We also demonstrate that good-quality ocean fluorescence line height retrievals can be achieved with GOME-2, SCIAMACHY, and similar instruments by utilizing the full complement of radiance measurements that span the red SIF emission feature.

New Methods for Retrieval of Chlorophyll Red Fluorescence from Hyperspectral Satellite Instruments: Simulations and Application to GOME-2 and SCIAMACHY

NASA Technical Reports Server (NTRS)

Joiner, Joanna; Yoshida, Yasuko; Guanter, Luis; Middleton, Elizabeth M.

2016-01-01

Global satellite measurements of solar-induced fluorescence (SIF) from chlorophyll over land and ocean have proven useful for a number of different applications related to physiology, phenology, and productivity of plants and phytoplankton. Terrestrial chlorophyll fluorescence is emitted throughout the red and far-red spectrum, producing two broad peaks near 683 and 736nm. From ocean surfaces, phytoplankton fluorescence emissions are entirely from the red region (683nm peak). Studies using satellite-derived SIF over land have focused almost exclusively on measurements in the far red (wavelengths greater than 712nm), since those are the most easily obtained with existing instrumentation. Here, we examine new ways to use existing hyperspectral satellite data sets to retrieve red SIF (wavelengths less than 712nm) over both land and ocean. Red SIF is thought to provide complementary information to that from the far red for terrestrial vegetation. The satellite instruments that we use were designed to make atmospheric trace-gas measurements and are therefore not optimal for observing SIF; they have coarse spatial resolution and only moderate spectral resolution (0.5nm). Nevertheless, these instruments, the Global Ozone Monitoring Instrument 2 (GOME-2) and the SCanning Imaging Absorption spectroMeter for Atmospheric CHartographY (SCIAMACHY), offer a unique opportunity to compare red and far-red terrestrial SIF at regional spatial scales. Terrestrial SIF has been estimated with ground-, aircraft-, or satellite-based instruments by measuring the filling-in of atmospheric andor solar absorption spectral features by SIF. Our approach makes use of the oxygen (O2) gamma band that is not affected by SIF. The SIF-free O2 gamma band helps to estimate absorption within the spectrally variable O2 B band, which is filled in by red SIF. SIF also fills in the spectrally stable solar Fraunhofer lines (SFLs) at wavelengths both inside and just outside the O2 B band, which further helps to estimate red SIF emission. Our approach is then an extension of previous approaches applied to satellite data that utilized only the filling-in of SFLs by red SIF. We conducted retrievals of red SIF using an extensive database of simulated radiances covering a wide range of conditions. Our new algorithm produces good agreement between the simulated truth and retrievals and shows the potential of the O2 bands for noise reduction in red SIF retrievals as compared with approaches that rely solely on SFL filling. Biases seen with existing satellite data, most likely due to instrumental artifacts that vary in time, space, and with instrument, must be addressed in order to obtain reasonable results. Our 8-year record of red SIF observations over land with the GOME-2 allows for the first time reliable global mapping of monthly anomalies. These anomalies are shown to have similar spatiotemporal structure as those in the far red, particularly for drought-prone regions. There is a somewhat larger percentage response in the red as compared with the far red for these areas that are drought sensitive. We also demonstrate that good-quality ocean fluorescence line height retrievals can be achieved with GOME-2, SCIAMACHY, and similar instruments by utilizing the full complement of radiance measurements that span the red SIF emission feature.

Microwave-Assisted Synthesis of Red-Light Emitting Au Nanoclusters with the Use of Egg White

ERIC Educational Resources Information Center

Tian, Jinghan; Yan, Lei; Sang, Aohua; Yuan, Hongyan; Zheng, Baozhan; Xiao, Dan

2014-01-01

We developed a simple, cost-effective, and eco-friendly method to synthesize gold nanoclusters (AuNCs) with red fluorescence. The experiment was performed using HAuCl[subscript 4], egg white, Na[subscript 2]CO[subscript 3] (known as soda ash or washing soda), and a microwave oven. In our experiment, fluorescent AuNCs were prepared within a…

Evaluation of acridine orange, LysoTracker Red, and quinacrine as fluorescent probes for long-term tracking of acidic vesicles.

PubMed

Pierzyńska-Mach, Agnieszka; Janowski, Paweł A; Dobrucki, Jurek W

2014-08-01

Acidic vesicles can be imaged and tracked in live cells after staining with several low molecular weight fluorescent probes, or with fluorescently labeled proteins. Three fluorescent dyes, acridine orange, LysoTracker Red DND-99, and quinacrine, were evaluated as acidic vesicle tracers for confocal fluorescence imaging and quantitative analysis. The stability of fluorescent signals, achievable image contrast, and phototoxicity were taken into consideration. The three tested tracers exhibit different advantages and pose different problems in imaging experiments. Acridine orange makes it possible to distinguish acidic vesicles with different internal pH but is fairly phototoxic and can cause spectacular bursts of the dye-loaded vesicles. LysoTracker Red is less phototoxic but its rapid photobleaching limits the range of useful applications considerably. We demonstrate that quinacrine is most suitable for long-term imaging when a high number of frames is required. This capacity made it possible to trace acidic vesicles for several hours, during a process of drug-induced apoptosis. An ability to record the behavior of acidic vesicles over such long periods opens a possibility to study processes like autophagy or long-term effects of drugs on endocytosis and exocytosis. © 2014 International Society for Advancement of Cytometry.

Fish with red fluorescent eyes forage more efficiently under dim, blue-green light conditions.

PubMed

Harant, Ulrike Katharina; Michiels, Nicolaas Karel

2017-04-20

Natural red fluorescence is particularly conspicuous in the eyes of some small, benthic, predatory fishes. Fluorescence also increases in relative efficiency with increasing depth, which has generated speculation about its possible function as a "light organ" to detect cryptic organisms under bluish light. Here we investigate whether foraging success is improved under ambient conditions that make red fluorescence stand out more, using the triplefin Tripterygion delaisi as a model system. We repeatedly presented 10 copepods to individual fish (n = 40) kept under a narrow blue-green spectrum and compared their performance with that under a broad spectrum with the same overall brightness. The experiment was repeated for two levels of brightness, a shaded one representing 0.4% of the light present at the surface and a heavily shaded one with about 0.01% of the surface brightness. Fish were 7% more successful at catching copepods under the narrow, fluorescence-friendly spectrum than under the broad spectrum. However, this effect was significant under the heavily shaded light treatment only. This outcome corroborates previous predictions that fluorescence may be an adaptation to blue-green, heavily shaded environments, which coincides with the opportunistic biology of this species that lives in the transition zone between exposed and heavily shaded microhabitats.

A PDMS-based cylindrical hybrid lens for enhanced fluorescence detection in microfluidic systems.

PubMed

Lin, Bor-Shyh; Yang, Yu-Ching; Ho, Chong-Yi; Yang, Han-Yu; Wang, Hsiang-Yu

2014-02-13

Microfluidic systems based on fluorescence detection have been developed and applied for many biological and chemical applications. Because of the tiny amount of sample in the system; the induced fluorescence can be weak. Therefore, most microfluidic systems deploy multiple optical components or sophisticated equipment to enhance the efficiency of fluorescence detection. However, these strategies encounter common issues of complex manufacturing processes and high costs. In this study; a miniature, cylindrical and hybrid lens made of polydimethylsiloxane (PDMS) to improve the fluorescence detection in microfluidic systems is proposed. The hybrid lens integrates a laser focusing lens and a fluorescence collecting lens to achieve dual functions and simplify optical setup. Moreover, PDMS has advantages of low-cost and straightforward fabrication compared with conventional optical components. The performance of the proposed lens is first examined with two fluorescent dyes and the results show that the lens provides satisfactory enhancement for fluorescence detection of Rhodamine 6G and Nile Red. The overall increments in collected fluorescence signal and detection sensitivity are more than 220% of those without lens, and the detection limits of Rhodamine 6G and Nile red are lowered to 0.01 μg/mL and 0.05 μg/mL, respectively. The hybrid lens is further applied to the detection of Nile red-labeled Chlorella vulgaris cells and it increases both signal intensity and detection sensitivity by more than 520%. The proposed hybrid lens also dramatically reduces the variation in detected signal caused by the deviation in incident angle of excitation light.

Characterization of novel bacteriochlorophyll-a-containing red filaments from alkaline hot springs in Yellowstone National Park.

PubMed

Boomer, S M; Pierson, B K; Austinhirst, R; Castenholz, R W

2000-09-01

Novel red, filamentous, gliding bacteria formed deep red layers in several alkaline hot springs in Yellowstone National Park. Filaments contained densely layered intracellular membranes and bacteriochlorophyll a. The in vivo absorption spectrum of the red layer filaments was distinct from other phototrophs, with unusual bacteriochlorophyll a signature peaks in the near-infrared (IR) region (807 nm and 911 nm). These absorption peaks were similar to the wavelengths penetrating to the red layer of the mats as measured with in situ spectroradiometry. The filaments also demonstrated maximal photosynthetic uptake of radiolabeled carbon sources at these wavelengths. The red layer filaments displayed anoxygenic photoheterotrophy, as evidenced by the specific incorporation of acetate, not bicarbonate, and by the absence of oxygen production. Photoheterotrophy was unaffected by sulfide and oxygen, but was diminished by high-intensity visible light. Near-IR radiation supported photoheterotrophy. Morphologically and spectrally similar filaments were observed in several springs in Yellowstone National Park, including Octopus Spring. Taken together, these data suggest that the red layer filaments are most similar to the photoheterotroph, Heliothrix oregonensis. Notable differences include mat position and coloration, absorption spectra, and prominent intracellular membranes.

Predicting Key Agronomic Soil Properties with UV-Vis Fluorescence Measurements Combined with Vis-NIR-SWIR Reflectance Spectroscopy: A Farm-Scale Study in a Mediterranean Viticultural Agroecosystem.

PubMed

Vaudour, Emmanuelle; Cerovic, Zoran G; Ebengo, Dav M; Latouche, Gwendal

2018-04-10

For adequate crop and soil management, rapid and accurate techniques for monitoring soil properties are particularly important when a farmer starts up his activities and needs a diagnosis of his cultivated fields. This study aimed to evaluate the potential of fluorescence measured directly on 146 whole soil solid samples, for predicting key soil properties at the scale of a 6 ha Mediterranean wine estate with contrasting soils. UV-Vis fluorescence measurements were carried out in conjunction with reflectance measurements in the Vis-NIR-SWIR range. Combining PLSR predictions from Vis-NIR-SWIR reflectance spectra and from a set of fluorescence signals enabled us to improve the power of prediction of a number of key agronomic soil properties including SOC, N tot , CaCO₃, iron, fine particle-sizes (clay, fine silt, fine sand), CEC, pH and exchangeable Ca 2+ with cross-validation RPD ≥ 2 and R² ≥ 0.75, while exchangeable K⁺, Na⁺, Mg 2+ , coarse silt and coarse sand contents were fairly predicted (1.42 ≤ RPD < 2 and 0.54 ≤ R² < 0.75). Predictions of SOC, N tot , CaCO₃, iron contents, and pH were still good (RPD ≥ 1.8, R² ≥ 0.68) when using a single fluorescence signal or index such as SFR_R or FERARI, highlighting the unexpected importance of red excitations and indices derived from plant studies. The predictive ability of single fluorescence indices or original signals was very significant for topsoil: this is very important for a farmer who wishes to update information on soil nutrient for the purpose of fertility diagnosis and particularly nitrogen fertilization. These results open encouraging perspectives for using miniaturized fluorescence devices enabling red excitation coupled with red or far-red fluorescence emissions directly in the field.

Predicting Key Agronomic Soil Properties with UV-Vis Fluorescence Measurements Combined with Vis-NIR-SWIR Reflectance Spectroscopy: A Farm-Scale Study in a Mediterranean Viticultural Agroecosystem

PubMed Central

Vaudour, Emmanuelle; Cerovic, Zoran G.; Ebengo, Dav M.; Latouche, Gwendal

2018-01-01

For adequate crop and soil management, rapid and accurate techniques for monitoring soil properties are particularly important when a farmer starts up his activities and needs a diagnosis of his cultivated fields. This study aimed to evaluate the potential of fluorescence measured directly on 146 whole soil solid samples, for predicting key soil properties at the scale of a 6 ha Mediterranean wine estate with contrasting soils. UV-Vis fluorescence measurements were carried out in conjunction with reflectance measurements in the Vis-NIR-SWIR range. Combining PLSR predictions from Vis-NIR-SWIR reflectance spectra and from a set of fluorescence signals enabled us to improve the power of prediction of a number of key agronomic soil properties including SOC, Ntot, CaCO3, iron, fine particle-sizes (clay, fine silt, fine sand), CEC, pH and exchangeable Ca2+ with cross-validation RPD ≥ 2 and R² ≥ 0.75, while exchangeable K+, Na+, Mg2+, coarse silt and coarse sand contents were fairly predicted (1.42 ≤ RPD < 2 and 0.54 ≤ R² < 0.75). Predictions of SOC, Ntot, CaCO3, iron contents, and pH were still good (RPD ≥ 1.8, R² ≥ 0.68) when using a single fluorescence signal or index such as SFR_R or FERARI, highlighting the unexpected importance of red excitations and indices derived from plant studies. The predictive ability of single fluorescence indices or original signals was very significant for topsoil: this is very important for a farmer who wishes to update information on soil nutrient for the purpose of fertility diagnosis and particularly nitrogen fertilization. These results open encouraging perspectives for using miniaturized fluorescence devices enabling red excitation coupled with red or far-red fluorescence emissions directly in the field. PMID:29642640

Far-red light photoactivatable near-infrared fluorescent proteins engineered from a bacterial phytochrome.

PubMed

Piatkevich, Kiryl D; Subach, Fedor V; Verkhusha, Vladislav V

2013-01-01

The ability to modulate the fluorescence of optical probes can be used to enhance signal-to-noise ratios for imaging within highly autofluorescent environments, such as intact tissues and living organisms. Here, we report two bacteriophytochrome-based photoactivatable near-infrared fluorescent proteins, named PAiRFP1 and PAiRFP2. PAiRFPs utilize haem-derived biliverdin, ubiquitous in mammalian tissues, as the chromophore. Initially weakly fluorescent PAiRFPs undergo photoconversion into a highly fluorescent state with excitation/emission at 690/717 nm following a brief irradiation with far-red light. After photoactivation, PAiRFPs slowly revert back to initial state, enabling multiple photoactivation-relaxation cycles. Low-temperature optical spectroscopy reveals several intermediates involved in PAiRFP photocycles, which all differ from that of the bacteriophytochrome precursor. PAiRFPs can be photoactivated in a spatially selective manner in mouse tissues, and optical modulation of their fluorescence allows for substantial contrast enhancement, making PAiRFPs advantageous over permanently fluorescent probes for in vivo imaging conditions of high autofluorescence and low signal levels.

Photoactivation mechanism of PAmCherry based on crystal structures of the protein in the dark and fluorescent states.

PubMed

Subach, Fedor V; Malashkevich, Vladimir N; Zencheck, Wendy D; Xiao, Hui; Filonov, Grigory S; Almo, Steven C; Verkhusha, Vladislav V

2009-12-15

Photoactivatable fluorescent proteins (PAFPs) are required for super-resolution imaging of live cells. Recently, the first red PAFP, PAmCherry1, was reported, which complements the photo-activatable GFP by providing a red super-resolution color. PAmCherry1 is originally "dark" but exhibits red fluorescence after UV-violet light irradiation. To define the structural basis of PAmCherry1 photoactivation, we determined its crystal structure in the dark and red fluorescent states at 1.50 A and 1.65 A, respectively. The non-coplanar structure of the chromophore in the dark PAmChery1 suggests the presence of an N-acylimine functionality and a single non-oxidized C(alpha)-C(beta) bond in the Tyr-67 side chain in the cyclized Met-66-Tyr-67-Gly-68 tripeptide. MS data of the chromophore-bearing peptide indicates the loss of 20 Da upon maturation, whereas tandem MS reveals the C(alpha)-N bond in Met-66 is oxidized. These data indicate that PAmCherry1 in the dark state possesses the chromophore N-[(E)-(5-hydroxy-1H-imidazol-2-yl)methylidene]acetamide, which, to our knowledge, has not been previously observed in PAFPs. The photoactivated PAmCherry1 exhibits a non-coplanar anionic DsRed-like chromophore but in the trans configuration. Based on the crystallographic analysis, MS data, and biochemical analysis of the PAmCherry1 mutants, we propose the detailed photoactivation mechanism. In this mechanism, the excited-state PAmCherry1 chromophore acts as the oxidant to release CO(2) molecule from Glu-215 via a Koble-like radical reaction. The Glu-215 decarboxylation directs the carbanion formation resulting in the oxidation of the Tyr-67 C(alpha)-C(beta) bond. The double bond extends the pi-conjugation between the phenolic ring of Tyr-67, the imidazolone, and the N-acylimine, resulting in the red fluorescent chromophore.

Fluorescence spectral analysis of resinite macerals from coals of the Hanna Formation, Wyoming, U.S.A.

USGS Publications Warehouse

Teerman, S.C.; Crelling, J.C.; Glass, G.B.

1987-01-01

Flourescence spectral analysis indicates that resinite macerals from Tertiary Hanna Formation coals (Hanna Coal Field, southcentral Wyoming, U.S.A.) can be separated into five distinct groups. The first resinite group fluoresces a a medium green (in blue light); its average spectral maximum occurs at or below 440 mm with a red/green quotient of 0.22. The second resinite group fluoresces yellow-green with an average spectral maximum of 500 nm and a red/green quotient of 0.53. The third resinite group displays a yellow fluorescence having an average spectral maximum of 580 nm and a red/green quotient of 0.86. The fourth resinite group fluorescence orange-brown having an average spectral maximum of 610 nm and a red/green quotient of 1.20. These four groups mostly occur as primary globular resinites exhibiting scratches and fractures, indicating that they are brittle, solid substances. Primary cell-filling and secondary fracture-filling resinites also occur in these four groups. The fifth group only occurs as a secondary void-filling material and lacks evidence of br of brittle properties. It fluoresces a reddish-brown, has a spectral maximum at 690 nm, and a red/green quotient of 1.54. The fifth group has properties resembling exsudatinite. The five resinite groups can be separated on the basis of their nine spectral properties alone, without qualitative petrographic interpretation. The relative quantities of the five resinite groups vary among Hanna Formation coals. The origins of these five resinite groups are probably related to their botanical properties and pre- and post-depossitional conditions. Overall, Hanna Formation resinites have petrographic characteristics similar to other North American resinites; however, only four resinite groups have been distinguished in in certain coals from Utah and New Mexico (U.S.A.), and western Canada. ?? 1987.

Taking into Account Interelement Interference in X-Ray Fluorescence Analysis of Thin Two-Layer Ti/V Systems

NASA Astrophysics Data System (ADS)

Mashin, N. I.; Razuvaev, A. G.; Cherniaeva, E. A.; Gafarova, L. M.; Ershov, A. V.

2018-03-01

We propose a new method for determining the thickness of layers in x-ray fluorescence analysis of two-layer Ti/V systems, using easily fabricated standardized film layers obtained by sputter deposition of titanium on a polymer film substrate. We have calculated correction factors taking into account the level of attenuation for the intensity of the primary emission from the x-ray tube and the analytical line for the element of the bottom layer in the top layer, and the enhancement of the fluorescence intensity for the top layer by the emission of atoms in the bottom layer.

Synthesis of yellow and red fluorescent 1,3a,6a-triazapentalenes and the theoretical investigation of their optical properties† †Electronic supplementary information (ESI) available: the experimental details for the synthesis of the triazapentalenes and the fluorescent cell staining, the absorption and fluorescence spectra, and the 1H and 13C NMR spectra. Also given are the molecular orbitals, the natural charges, the dipole moments, and the Cartesian coordinates of the triazapentalenes (1a, 1b, 1g, 1e, and 1f). See DOI: 10.1039/c4sc02780a Click here for additional data file.

PubMed Central

Osawa, Ayumi; Mera, Akane; Tano, Fumi; Chuman, Yoshiro; Sakuda, Eri; Taketsugu, Tetsuya; Sakaguchi, Kazuyasu; Kitamura, Noboru

2015-01-01

To expand the originally developed fluorescent 1,3a,6a-triazapentalenes as fluorescent labelling reagents, the fluorescence wavelength of the 1,3a,6a-triazapentalenes was extended to the red color region. Based on the noteworthy correlation of the fluorescence wavelength with the inductive effect of the 2-substituent, electron-deficient 2-(2-cyano-4-methoxycarbonylphenyl)-1,3a,6a-triazapentalene and 2-(2,6-dicyano-4-methoxycarbonylphenyl)-1,3a,6a-triazapentalene were synthesized. The former exhibited yellow fluorescence and the latter exhibited red fluorescence, and both compounds exhibited large Stokes shifts, and the 1,3a,6a-triazapentalene system enabled the same fluorescent chromophore to cover the entire region of visible wavelengths. The potential applications of the 1,3a,6a-triazapentalenes as fluorescent probes in the fields of the life sciences were investigated, and the 1,3a,6a-triazapentalene system was clearly proven to be useful as a fluorescent reagent for live cell imaging. Quantum chemical calculations were performed to investigate the optical properties of the 1,3a,6a-triazapentalenes. These calculations revealed that the excitation involves a significant charge-transfer from the 1,3a,6a-triazapentalene skeleton to the 2-substituent. The calculated absorption and fluorescence wavelengths showed a good correlation with the experimental ones, and thus the system could enable the theoretical design of substituents with the desired optical properties. PMID:29560196

[Effects of white organic light-emitting devices using color conversion films on electroluminescence spectra].

PubMed

Hou, Qing-Chuan; Wu, Xiao-Ming; Hua, Yu-Lin; Qi, Qing-Jin; Li, Lan; Yin, Shou-Gen

2010-06-01

The authors report a novel white organic light-emitting device (WOLED), which uses a strategy of exciting organic/ inorganic color conversion film with a blue organic light-emitting diode (OLED). The luminescent layer of the blue OLED was prepared by use of CBP host blended with a blue highly fluorescent dye N-BDAVBi. The organic/inorganic color conversion film was prepared by dispersing a mixture of red pigment VQ-D25 and YAG : Ce3+ phosphor in PMMA. The authors have achieved a novel WOLED with the high color stability by optimizing the thickness and fluorescent pigment concentration of the color conversion film. When the driving voltage varied between 6 and 14 V, the color coordinates (CIE) varied slightly from (0.354, 0.304) to (0.357, 0.312) and the maximum current efficiency is about 5.8 cd x A(-1) (4.35 mA x cm(-2)), the maximum brightness is 16 800 cd x m(-2) at the operating voltage of 14 V.

All-optical recording and stimulation of retinal neurons in vivo in retinal degeneration mice

PubMed Central

Strazzeri, Jennifer M.; Williams, David R.; Merigan, William H.

2018-01-01

Here we demonstrate the application of a method that could accelerate the development of novel therapies by allowing direct and repeatable visualization of cellular function in the living eye, to study loss of vision in animal models of retinal disease, as well as evaluate the time course of retinal function following therapeutic intervention. We use high-resolution adaptive optics scanning light ophthalmoscopy to image fluorescence from the calcium sensor GCaMP6s. In mice with photoreceptor degeneration (rd10), we measured restored visual responses in ganglion cell layer neurons expressing the red-shifted channelrhodopsin ChrimsonR over a six-week period following significant loss of visual responses. Combining a fluorescent calcium sensor, a channelrhodopsin, and adaptive optics enables all-optical stimulation and recording of retinal neurons in the living eye. Because the retina is an accessible portal to the central nervous system, our method also provides a novel non-invasive method of dissecting neuronal processing in the brain. PMID:29596518

A novel dicyanoisophorone based red-emitting fluorescent probe with a large Stokes shift for detection of hydrazine in solution and living cells

NASA Astrophysics Data System (ADS)

Lv, Hongshui; Sun, Haiyan; Wang, Shoujuan; Kong, Fangong

2018-05-01

A novel dicyanoisophorone based fluorescent probe HP was developed to detect hydrazine. Upon the addition of hydrazine, probe HP displayed turn-on fluorescence in the red region with a large Stokes shift (180 nm). This probe exhibited high selectivity and high sensitivity to hydrazine in solution. The detection limit of HP was found to be 3.26 ppb, which was lower than the threshold limit value set by USEPA (10 ppb). Moreover, the probe was successfully applied to detect hydrazine in different water samples and living cells.

Photoproduct formation of endogeneous protoporphyrin and its photodynamic activity

NASA Astrophysics Data System (ADS)

Koenig, Karsten; Schneckenburger, Herbert; Rueck, Angelika C.; Auchter, S.

1991-11-01

Human skin shows a strong autofluorescence in the red spectral region caused on the porphyrin production of the Gram positive lipophile skin bacterium Propionibacterium acnes. Irradiation of these bacteria reduces the integral fluorescence intensity and induces the formation of fluorescent photoproducts. The fluorescence band at around 670 nm and the decay times of around 1 ns and 5 ns are typical for protoporphyrin products. The photoproduct formation is connected with an increased absorption in the red spectral region. However the photodynamic activity of these photoproducts determined by scattering measurements on human erythrocytes is lower than that of protoporphyrin IX. 1:

Light adaptation of the unicellular red alga, Cyanidioschyzon merolae, probed by time-resolved fluorescence spectroscopy.

PubMed

Ueno, Yoshifumi; Aikawa, Shimpei; Kondo, Akihiko; Akimoto, Seiji

2015-08-01

Photosynthetic organisms change the quantity and/or quality of their pigment-protein complexes and the interactions among these complexes in response to light conditions. In the present study, we analyzed light adaptation of the unicellular red alga Cyanidioschyzon merolae, whose pigment composition is similar to that of cyanobacteria because its phycobilisomes (PBS) lack phycoerythrin. C. merolae were grown under different light qualities, and their responses were measured by steady-state absorption, steady-state fluorescence, and picosecond time-resolved fluorescence spectroscopies. Cells were cultivated under four monochromatic light-emitting diodes (blue, green, yellow, and red), and changes in pigment composition and energy transfer were observed. Cells grown under blue and green light increased their relative phycocyanin levels compared with cells cultured under white light. Energy-transfer processes to photosystem I (PSI) were sensitive to yellow and red light. The contribution of direct energy transfer from PBS to PSI increased only under yellow light, while red light induced a reduction in energy transfer from photosystem II to PSI and an increase in energy transfer from light-harvesting chlorophyll protein complex I to PSI. Differences in pigment composition, growth, and energy transfer under different light qualities are discussed.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Cotlet, M.; Mudalige, K.; Habuchi, S.

HcRed is a dimeric intrinsically fluorescent protein with origins in the sea anemone Heteractis crispa. This protein exhibits deep red absorption and emission properties. Using a combination of ensemble and single molecule methods and by varying environmental parameters such as temperature and pH, we found spectroscopic evidence for the presence of two ground state conformers, trans and cis chromophores that are in thermal equilibrium and that follow different excited-state pathways upon exposure to light. The photocycle of HcRed appears to be a combination of both kindling proteins and bright emitting GFP/GFP-like proteins: the trans chromophore undergoes light driven isomerization followedmore » by radiative relaxation with a fluorescence lifetime of 0.5 ns. The cis chromophore exhibits a photocycle similar to bright GFPs and GFP-like proteins such as enhanced GFP, enhanced YFP or DsRed, with radiative relaxation with a fluorescence lifetime of 1.5 ns, singlet-triplet deactivation on a microsecond time scale and solvent controlled protonation/deprotonation in tens of microseconds. Using single molecule spectroscopy, we identify trans and cis conformers at the level of individual moieties and show that it is possible that the two conformers can coexist in a single protein due to the dimeric nature of HcRed.« less

Highly efficient red OLEDs using DCJTB as the dopant and delayed fluorescent exciplex as the host

PubMed Central

Zhao, Bo; Zhang, Tianyou; Chu, Bei; Li, Wenlian; Su, Zisheng; Wu, Hairuo; Yan, Xingwu; Jin, Fangming; Gao, Yuan; Liu, Chengyuan

2015-01-01

In this manuscript, we demonstrated a highly efficient DCJTB emission with delayed fluorescent exciplex TCTA:3P-T2T as the host. For the 1.0% DCJTB doped concentration, a maximum luminance, current efficiency, power efficiency and EQE of 22,767 cd m−2, 22.7 cd A−1, 21.5 lm W−1 and 10.15% were achieved, respectively. The device performance is the best compared to either red OLEDs with traditional fluorescent emitter or traditional red phosphor of Ir(piq)3 doped into CBP host. The extraction of so high efficiency can be explained as the efficient triplet excitons up-conversion of TCTA:3P-T2T and the energy transfer from exciplex host singlet state to DCJTB singlet state. PMID:26023882

Fluorescence emission of disperse Red 1 in solution at room temperature.

PubMed

Toro, Carlos; Thibert, Arthur; De Boni, Leonardo; Masunov, Artëm E; Hernández, Florencio E

2008-01-24

In this article, we report the fluorescence emission of Disperse Red 1 in solution at room temperature and pumping at 532 nm with a 25 mW diode laser. We have measured its fluorescence quantum yield in methanol, ethylene glycol, glycerol, and phenol obtaining values as high as 10(-3) in the aliphatic alcohols. The excitation spectra of Disperse Red 1 in all four solvents as well as its excitation anisotropy in glycerol are presented. Applying a Gaussian decomposition method to the absorption spectra along with the support from the excitation spectra, the positions of the different transitions in this pseudo-stilbene azobenzene dye were determined. Solvatochromic and isomerization constraint effects are discussed. Calculations using density functional theory at TD-B3LYP/6-31G*//HF/6-31G* level were performed to interpret the experimental observations.

Accurate thermometry based on the red and green fluorescence intensity ratio in NaYF₄: Yb, Er nanocrystals for bioapplication.

PubMed

Liu, Lixin; Qin, Feng; Lv, Tianquan; Zhang, Zhiguo; Cao, Wenwu

2016-10-15

A biological temperature measurement method based on the fluorescence intensity ratio (FIR) was developed to reduce uncertainty. The upconversion luminescence of NaYF₄:Yb, Er nanocrystals was studied as a function of temperature around the physiologically relevant range of 300-330 K. We found that the green-green FIR Fe and red-green FIR (I₆₆₀/I₅₄₀) varied linearly as temperature increased. The thermometric uncertainties using the two FIRs were discussed and were determined to be almost constant at 0.6 and 0.09 K for green-green and red-green, respectively. The lower thermometric uncertainty comes from the intense signal-to-noise ratio of the measured FIRs owing to their comparable fluorescence intensities.

A simple and sensitive fluorescence method for the determination of trace ozone in air using acridine red as a probe.

PubMed

Liu, Qingye; Lin, Chenyin; Zhang, Xinghui; Wen, Guiqing; Liang, Aihui

2014-12-01

The ozone in an air sample was trapped by H3 BO3 -LK solution to produce iodine (I2) that interacted with excess I(-) to form I3(-). In pH 4.0 acetate buffer solutions, the I3(-) reacted with acridine red to form acridine red-I3 ion association particles that resulted in the fluorescence peak decreased at 553 nm. The decreased value ΔF553 nm is linear to the O3 concentration in the range 0.08-53.3 × 10(-6) mol/L, with a detection limit of 4 × 10(-8) mol/L. This fluorescence method was used to determine ozone in air samples, and the results were in agreement with that of indigo carmine spectrophotometry. Copyright © 2014 John Wiley & Sons, Ltd.

Depth-Penetrating Measurements Developed for Thermal Barrier Coatings Incorporating Thermographic Phosphors

NASA Technical Reports Server (NTRS)

Eldridge, Jeffrey I.; Bencic, Timothy J.

2004-01-01

The insulating properties of thermal barrier coatings (TBCs) provide highly beneficial thermal protection to turbine engine components by reducing the temperature sustained by those components. Therefore, measuring the temperature beneath the TBC is critical for determining whether the TBC is performing its insulating function. Currently, noncontact temperature measurements are performed by infrared pyrometry, which unfortunately measures the TBC surface temperature rather than the temperature of the underlying component. To remedy this problem, the NASA Glenn Research Center, under the Information Rich Test Instrumentation Project, developed a technique to measure the temperature beneath the TBC by incorporating a thin phosphor layer beneath the TBC. By performing fluorescence decay-time measurements on light emission from this phosphor layer, Glenn successfully measured temperatures from the phosphor layer up to 1100 C. This is the first successful demonstration of temperature measurements that penetrate beneath the TBC. Thermographic phosphors have a history of providing noncontact surface temperature measurements. Conventionally, a thermographic phosphor is applied to the material surface and temperature measurements are performed by exciting the phosphor with ultraviolet light and then measuring the temperature-dependent decay time of the phosphor emission at a longer wavelength. The innovative feature of the new approach is to take advantage of the relative transparency of the TBC (composed of yttria-stabilized zirconia) in order to excite and measure the phosphor emission beneath the TBC. The primary obstacle to achieving depth-penetrating temperature measurements is that the TBCs are completely opaque to the ultraviolet light usually employed to excite the phosphor. The strategy that Glenn pursued was to select a thermographic phosphor that could be excited and emit at wavelengths that could be transmitted through the TBC. The phosphor that was selected was yttria doped with europia (Y2O3:Eu), which has a minor excitation peak at 532 nm (green) and an emission peak at 611 nm (red)--both are wavelengths that exhibit significant transmission through the TBC. The measurements were performed on specimens consisting of a 25- m-thick phosphor layer beneath a 100- m-thick TBC. The 532-nm (green) excitation light was provided by a frequency-doubled YAG:Nd (yttrium-aluminum-garnet:neodymium) laser, and the fluorescence decay time measurements were acquired with a modified Raman microscope. The preceding graph compares the intensity of the phosphor emission of the phosphor layer above the TBC versus that of the phosphor layer beneath the TBC. Although there was considerable attenuation of the phosphor signal (a factor of 30), the phosphor emission at the reduced intensity was more than sufficient to perform fluorescence decay time measurements. The following graph shows the fluorescence lifetime temperature dependency for the Y2O3:Eu phosphor layers both above and below the TBC. These curves show an excellent match and indicate that, despite the attenuation due to the overlying TBC, the phosphor layer beneath the TBC still functions as an effective temperature indicator.

Magnetic Iron Oxide Nanoparticles and a Polydiacetylene Coating to Create a Biocompatible and Stable Molecule for Use in Cancer Diagnostics and Early Detection in Molecular Medicine

NASA Astrophysics Data System (ADS)

Bhatnagar, Shweta

Earlier cancer detection and diagnosis is essential to prevent cancer mortality in nanomedicine and nanotechnology. Fluorescence and magnetic signals provide a way for earlier detection through imaging systems. Magnetic iron oxide nanoparticles have a superparamagnetism feature that allows them to act as contrast agents that can be detected through a magnetic resonance imaging system. These iron oxide cores have a polymer coating around them to provide stability, prevent aggregation, and allow for biocompatibility within the body. In addition, these functional coatings can have ligands and peptides for detection and therapy purposes. One functional coating is a polydiacetylene coating due to its chromatic and optical properties. When polymerized, it has the ability to change color in the visible spectrum to blue (not a fluorescent signal) and when heated, it changes to a red color (fluorescent signal). This way a strong and stable layer is formed around the iron oxide cores. These coatings are placed on the iron cores using a modified dual solvent exchange method, in which DMSO is slowly replaced by water without the use of organic solvents previous used. In addition, these nanoparticles can then be PEGylated, which provides a more stable and water soluble compound in aqueous solutions. Measurements can be taken through dynamic light scattering for size distributions and zeta potential and the Nanodrop for absorbance. Ideal sizes are about 30 nm for MNPs. Moreover, for future directions, there can be more molecules attached to the coated layers to use for molecular detection and analysis.

A novel far-red fluorescent xenograft model of ovarian carcinoma for preclinical evaluation of HER2-targeted immunotoxins

PubMed Central

Zdobnova, Tatiana; Sokolova, Evgeniya; Stremovskiy, Oleg; Karpenko, Dmitry; Telford, William; Turchin, Ilya; Balalaeva, Irina; Deyev, Sergey

2015-01-01

We have created a novel fluorescent model of a human ovarian carcinoma xenograft overexpressing receptor HER2, a promising molecular target of solid tumors. The model is based on a newly generated SKOV-kat cell line stably expressing far-red fluorescent protein Katushka. Katushka is most suitable for the in vivo imaging due to an optimal combination of high brightness and emission in the “window of tissue transparency”. The relevance of the fluorescent model for the in vivo monitoring of tumor growth and response to treatment was demonstrated using a newly created HER2-targeted recombinant immunotoxin based on the 4D5scFv antibody and a fragment of the Pseudomonas exotoxin A. PMID:26436696

Multiexcitation Fluorogenic Labeling of Surface, Intracellular, and Total Protein Pools in Living Cells

PubMed Central

2016-01-01

Malachite green (MG) is a fluorogenic dye that shows fluorescence enhancement upon binding to its engineered cognate protein, a fluorogen activating protein (FAP). Energy transfer donors such as cyanine and rhodamine dyes have been conjugated with MG to modify the spectral properties of the fluorescent complexes, where the donor dyes transfer energy through Förster resonance energy transfer to the MG complex resulting in binding-conditional fluorescence emission in the far-red region. In this article, we use a violet-excitable dye as a donor to sensitize the far-red emission of the MG-FAP complex. Two blue emitting fluorescent coumarin dyes were coupled to MG and evaluated for energy transfer to the MG-FAP complex via its secondary excitation band. 6,8-Difluoro-7-hydroxycoumarin-3-carboxylic acid (Pacific blue, PB) showed the most efficient energy transfer and maximum brightness in the far-red region upon violet (405 nm) excitation. These blue-red (BluR) tandem dyes are spectrally varied from other tandem dyes and are able to produce fluorescence images of the MG-FAP complex with a large Stokes shift (>250 nm). These dyes are cell-permeable and are used to label intracellular proteins. Used together with a cell-impermeable hexa-Cy3-MG (HCM) dye that labels extracellular proteins, we are able to visualize extracellular, intracellular, and total pools of cellular protein using one fluorogenic tag that combines with distinct dyes to effect different spectral characteristics. PMID:27159569

Red Fluorescent Proteins for Gene Expression and Protein Localization Studies in Streptococcus pneumoniae and Efficient Transformation with DNA Assembled via the Gibson Assembly Method.

PubMed

Beilharz, Katrin; van Raaphorst, Renske; Kjos, Morten; Veening, Jan-Willem

2015-10-01

During the last decades, a wide range of fluorescent proteins (FPs) have been developed and improved. This has had a great impact on the possibilities in biological imaging and the investigation of cellular processes at the single-cell level. Recently, we have benchmarked a set of green fluorescent proteins (GFPs) and generated a codon-optimized superfolder GFP for efficient use in the important human pathogen Streptococcus pneumoniae and other low-GC Gram-positive bacteria. In the present work, we constructed and compared four red fluorescent proteins (RFPs) in S. pneumoniae. Two orange-red variants, mOrange2 and TagRFP, and two far-red FPs, mKate2 and mCherry, were codon optimized and examined by fluorescence microscopy and plate reader assays. Notably, protein fusions of the RFPs to FtsZ were constructed by direct transformation of linear Gibson assembly (isothermal assembly) products, a method that speeds up the strain construction process significantly. Our data show that mCherry is the fastest-maturing RFP in S. pneumoniae and is best suited for studying gene expression, while mKate2 and TagRFP are more stable and are the preferred choices for protein localization studies. The RFPs described here will be useful for cell biology studies that require multicolor labeling in S. pneumoniae and related organisms. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Sorting Out Antibiotics' Mechanisms of Action: a Double Fluorescent Protein Reporter for High-Throughput Screening of Ribosome and DNA Biosynthesis Inhibitors

PubMed Central

Osterman, Ilya A.; Komarova, Ekaterina S.; Shiryaev, Dmitry I.; Korniltsev, Ilya A.; Khven, Irina M.; Lukyanov, Dmitry A.; Tashlitsky, Vadim N.; Serebryakova, Marina V.; Efremenkova, Olga V.; Ivanenkov, Yan A.; Bogdanov, Alexey A.; Dontsova, Olga A.

2016-01-01

In order to accelerate drug discovery, a simple, reliable, and cost-effective system for high-throughput identification of a potential antibiotic mechanism of action is required. To facilitate such screening of new antibiotics, we created a double-reporter system for not only antimicrobial activity detection but also simultaneous sorting of potential antimicrobials into those that cause ribosome stalling and those that induce the SOS response due to DNA damage. In this reporter system, the red fluorescent protein gene rfp was placed under the control of the SOS-inducible sulA promoter. The gene of the far-red fluorescent protein, katushka2S, was inserted downstream of the tryptophan attenuator in which two tryptophan codons were replaced by alanine codons, with simultaneous replacement of the complementary part of the attenuator to preserve the ability to form secondary structures that influence transcription termination. This genetically modified attenuator makes possible Katushka2S expression only upon exposure to ribosome-stalling compounds. The application of red and far-red fluorescent proteins provides a high signal-to-background ratio without any need of enzymatic substrates for detection of the reporter activity. This reporter was shown to be efficient in high-throughput screening of both synthetic and natural chemicals. PMID:27736765

Spatially Controlled Fabrication of Brightly Fluorescent Nanodiamond-Array with Enhanced Far-Red Si-V Luminescence

PubMed Central

Singh, Sonal; Thomas, Vinoy; Martyshkin, Dmitry; Kozlovskaya, Veronika; Kharlampieva, Eugenia

2014-01-01

We demonstrate a novel approach to precise pattern fluorescent nanodiamond-arrays with enhanced far-red intense photostable luminescence from silicon-vacancy (Si-V) defect centers. The precision-patterned pre-growth seeding of nanodiamonds is achieved by scanning probe “Dip-Pen” nanolithography technique using electrostatically-driven transfer of nanodiamonds from “inked” cantilevers to a UV-treated hydrophilic SiO2 substrate. The enhanced emission from nanodiamond-dots in the far-red is achieved by incorporating Si-V defect centers in subsequent chemical vapor deposition treatment. The development of a suitable nanodiamond ink, mechanism of ink transport, and effect of humidity, dwell time on nanodiamond patterning are investigated. The precision-patterning of as-printed (pre-CVD) arrays with dot diameter and dot height as small as 735 nm ± 27 nm, 61 nm ± 3 nm, respectively and CVD-treated fluorescent ND-arrays with consistently patterned dots having diameter and height as small as 820 nm ± 20 nm, 245 nm ± 23 nm, respectively using 1 s dwell time and 30% RH is successfully achieved. We anticipate that the far-red intense photostable luminescence (~738 nm) observed from Si-V defect centers integrated in spatially arranged nanodiamonds could be beneficial for the development of the next generation fluorescent based devices and applications. PMID:24394286

Improving spinach, radish, and lettuce growth under red light-emitting diodes (LEDs) with blue light supplementation

NASA Technical Reports Server (NTRS)

Yorio, N. C.; Goins, G. D.; Kagie, H. R.; Wheeler, R. M.; Sager, J. C.

2001-01-01

Radish (Raphanus sativus L. cv. Cherriette), lettuce (Lactuca sativa L. cv. Waldmann's Green), and spinach (Spinacea oleracea L. cv. Nordic IV) plants were grown under 660-nm red light-emitting diodes (LEDs) and were compared at equal photosynthetic photon flux (PPF) with either plants grown under cool-white fluorescent lamps (CWF) or red LEDs supplemented with 10% (30 micromoles m-2 s-1) blue light (400-500 nm) from blue fluorescent (BF) lamps. At 21 days after planting (DAP), leaf photosynthetic rates and stomatal conductance were greater for plants grown under CWF light than for those grown under red LEDs, with or without supplemental blue light. At harvest (21 DAP), total dry-weight accumulation was significantly lower for all species tested when grown under red LEDs alone than when grown under CWF light or red LEDs + 10% BF light. Moreover, total dry weight for radish and spinach was significantly lower under red LEDs + 10% BF than under CWF light, suggesting that addition of blue light to the red LEDs was still insufficient for achieving maximal growth for these crops.

In situ measurement of airway surface liquid [K+] using a ratioable K+-sensitive fluorescent dye.

PubMed

Namkung, Wan; Song, Yuanlin; Mills, Aaron D; Padmawar, Prashant; Finkbeiner, Walter E; Verkman, A S

2009-06-05

The airway surface liquid (ASL) is the thin fluid layer lining airway surface epithelial cells, whose volume and composition are tightly regulated and may be abnormal in cystic fibrosis (CF). We synthesized a two-color fluorescent dextran to measure ASL [K(+)], TAC-Lime-dextran-TMR, consisting of a green-fluorescing triazacryptand K(+) ionophore-Bodipy conjugate, coupled to dextran, together with a red fluorescing tetramethylrhodamine reference chromophore. TAC-Lime-dextran-TMR fluorescence was K(+)-selective, increasing >4-fold with increasing [K(+)] from 0 to 40 mm. In well differentiated human airway epithelial cells, ASL [K(+)] was 20.8 +/- 0.3 mm and decreased by inhibition of the Na(+)/K(+) pump (ouabain), ENaC (amiloride), CF transmembrane conductance regulator (CFTR(inh)-172), or K(+) channels (TEA or XE991). ASL [K(+)] was increased by forskolin but not affected by Na(+)/K(+)/2Cl(-) cotransporter inhibition (bumetanide). Functional and expression studies indicated the involvement of [K(+)] channels KCNQ1, KCNQ3, and KCNQ5 as determinants of ASL [K(+)]. [K(+)] in CF cultures was similar to that in non-CF cultures, suggesting that abnormal ASL [K(+)] is not a factor in CF lung disease. In intact airways, ASL [K(+)] was also well above extracellular [K(+)]: 22 +/- 1 mm in pig trachea ex vivo and 16 +/- 1 mm in mouse trachea in vivo. Our results provide the first noninvasive measurements of [K(+)] in the ASL and indicate the involvement of apical and basolateral membrane ion transporters in maintaining a high ASL [K(+)].

Deep in vivo two-photon microscopy with a low cost custom built mode-locked 1060 nm fiber laser

PubMed Central

Perillo, Evan P.; McCracken, Justin E.; Fernée, Daniel C.; Goldak, John R.; Medina, Flor A.; Miller, David R.; Yeh, Hsin-Chih; Dunn, Andrew K.

2016-01-01

Here we demonstrate that a mode-locked ytterbium fiber laser for two-photon fluorescence microscopy can be built for $13,000. The laser emits at a wavelength of 1060 nm with a usable average power of 1 W at a repetition rate of 40 MHz and a compressed pulse width of 81 fs at the sample. The laser is used to obtain deep in vivo two-color images of layer-V pyramidal neurons expressing YFP and vasculature labelled with Texas Red at depths up to 900 µm. The sub-1 µm features of dendritic spines can be resolved at a 200 µm depth. PMID:26977343

Phenol red-silk tyrosine cross-linked hydrogels.

PubMed

Sundarakrishnan, Aswin; Herrero Acero, Enrique; Coburn, Jeannine; Chwalek, Karolina; Partlow, Benjamin; Kaplan, David L

2016-09-15

Phenol red is a cytocompatible pH sensing dye that is commonly added to cell culture media, but removed from some media formulations due to its structural mimicry of estrogen. Phenol red free media is also used during live cell imaging, to avoid absorbance and fluorescence quenching of fluorophores. To overcome these complications, we developed cytocompatible and degradable phenol red-silk tyrosine cross-linked hydrogels using horseradish peroxidase (HRP) enzyme and hydrogen peroxide (H2O2). Phenol red added to silk during tyrosine crosslinking accelerated di-tyrosine formation in a concentration-dependent reaction. Phenol red diffusion studies and UV-Vis spectra of phenol red-silk tyrosine hydrogels at different pHs showed altered absorption bands, confirming entrapment of dye within the hydrogel network. LC-MS of HRP-reacted phenol red and N-acetyl-l-tyrosine reaction products confirmed covalent bonds between the phenolic hydroxyl group of phenol red and tyrosine on the silk. At lower phenol red concentrations, leak-proof hydrogels which did not release phenol red were fabricated and found to be cytocompatible based on live-dead staining and alamar blue assessments of encapsulated fibroblasts. Due to the spectral overlap between phenol red absorbance at 415nm and di-tyrosine fluorescence at 417nm, phenol red-silk hydrogels provide both absorbance and fluorescence-based pH sensing. With an average pKa of 6.8 and good cytocompatibiltiy, phenol red-silk hydrogels are useful for pH sensing in phenol red free systems, cellular microenvironments and bioreactors. Phenol red entrapped within hydrogels facilitates pH sensing in phenol red free environments. Leak-proof phenol red based pH sensors require covalent binding techniques, but are complicated due to the lack of amino or carboxyl groups on phenol red. Currently, there is no simple, reliable technique to covalently link phenol red to hydrogel matrices, for real-time pH sensing in cell culture environments. Herein, we take advantage of phenolic groups for covalent linkage of phenol red to silk tyrosine in the presence of HRP and H2O2. The novelty of the current system stems from its simplicity and the use of silk protein to create a cytocompatible, degradable sensor capable of real-time pH sensing in cell culture microenvironments. Copyright © 2016 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

The first tandem, all-exciplex-based WOLED.

PubMed

Hung, Wen-Yi; Fang, Guan-Cheng; Lin, Shih-Wei; Cheng, Shuo-Hsien; Wong, Ken-Tsung; Kuo, Ting-Yi; Chou, Pi-Tai

2014-06-04

Exploiting our recently developed bilayer interface methodology, together with a new wide energy-gap, low LUMO acceptor (A) and the designated donor (D) layers, we succeeded in fabricating an exciplex-based organic light-emitting diode (OLED) systematically tuned from blue to red. Further optimization rendered a record-high blue exciplex OLED with η(ext) of 8%. We then constructed a device structure configured by two parallel blend layers of mCP/PO-T2T and DTAF/PO-T2T, generating blue and yellow exciplex emission, respectively. The resulting device demonstrates for the first time a tandem, all-exciplex-based white-light OLED (WOLED) with excellent efficiencies η(ext): 11.6%, η(c): 27.7 cd A(-1), and η(p): 15.8 ml W(-1) with CIE(0.29, 0.35) and CRI 70.6 that are nearly independent of EL intensity. The tandem architecture and blend-layer D/A (1:1) configuration are two key elements that fully utilize the exciplex delay fluorescence, providing a paragon for the use of low-cost, abundant organic compounds en route to commercial WOLEDs.

The First Tandem, All-exciplex-based WOLED

NASA Astrophysics Data System (ADS)

Hung, Wen-Yi; Fang, Guan-Cheng; Lin, Shih-Wei; Cheng, Shuo-Hsien; Wong, Ken-Tsung; Kuo, Ting-Yi; Chou, Pi-Tai

2014-06-01

Exploiting our recently developed bilayer interface methodology, together with a new wide energy-gap, low LUMO acceptor (A) and the designated donor (D) layers, we succeeded in fabricating an exciplex-based organic light-emitting diode (OLED) systematically tuned from blue to red. Further optimization rendered a record-high blue exciplex OLED with ηext of 8%. We then constructed a device structure configured by two parallel blend layers of mCP/PO-T2T and DTAF/PO-T2T, generating blue and yellow exciplex emission, respectively. The resulting device demonstrates for the first time a tandem, all-exciplex-based white-light OLED (WOLED) with excellent efficiencies ηext: 11.6%, ηc: 27.7 cd A-1, and ηp: 15.8 ml W-1 with CIE(0.29, 0.35) and CRI 70.6 that are nearly independent of EL intensity. The tandem architecture and blend-layer D/A (1:1) configuration are two key elements that fully utilize the exciplex delay fluorescence, providing a paragon for the use of low-cost, abundant organic compounds en route to commercial WOLEDs.

The First Tandem, All-exciplex-based WOLED

PubMed Central

Hung, Wen-Yi; Fang, Guan-Cheng; Lin, Shih-Wei; Cheng, Shuo-Hsien; Wong, Ken-Tsung; Kuo, Ting-Yi; Chou, Pi-Tai

2014-01-01

Exploiting our recently developed bilayer interface methodology, together with a new wide energy-gap, low LUMO acceptor (A) and the designated donor (D) layers, we succeeded in fabricating an exciplex-based organic light-emitting diode (OLED) systematically tuned from blue to red. Further optimization rendered a record-high blue exciplex OLED with ηext of 8%. We then constructed a device structure configured by two parallel blend layers of mCP/PO-T2T and DTAF/PO-T2T, generating blue and yellow exciplex emission, respectively. The resulting device demonstrates for the first time a tandem, all-exciplex-based white-light OLED (WOLED) with excellent efficiencies ηext: 11.6%, ηc: 27.7 cd A−1, and ηp: 15.8 ml W−1 with CIE(0.29, 0.35) and CRI 70.6 that are nearly independent of EL intensity. The tandem architecture and blend-layer D/A (1:1) configuration are two key elements that fully utilize the exciplex delay fluorescence, providing a paragon for the use of low-cost, abundant organic compounds en route to commercial WOLEDs. PMID:24895098

Nile Red Detection of Bacterial Hydrocarbons and Ketones in a High-Throughput Format

DOE Office of Scientific and Technical Information (OSTI.GOV)

Pinzon, NM; Aukema, KG; Gralnick, JA

A method for use in high-throughput screening of bacteria for the production of long-chain hydrocarbons and ketones by monitoring fluorescent light emission in the presence of Nile red is described. Nile red has previously been used to screen for polyhydroxybutyrate (PHB) and fatty acid esters, but this is the first report of screening for recombinant bacteria making hydrocarbons or ketones. The microtiter plate assay was evaluated using wild-type and recombinant strains of Shewanella oneidensis and Escherichia coli expressing the enzyme OleA, previously shown to initiate hydrocarbon biosynthesis. The strains expressing exogenous Stenotrophomonas maltophilia oleA, with increased levels of ketone productionmore » as determined by gas chromatography-mass spectrometry, were distinguished with Nile red fluorescence. Confocal microscopy images of S. oneidensis oleA-expressing strains stained with Nile red were consistent with a membrane localization of the ketones. This differed from Nile red staining of bacterial PHB or algal lipid droplets that showed intracellular inclusion bodies. These results demonstrated the applicability of Nile red in a high-throughput technique for the detection of bacterial hydrocarbons and ketones. IMPORTANCE In recent years, there has been renewed interest in advanced biofuel sources such as bacterial hydrocarbon production. Previous studies used solvent extraction of bacterial cultures followed by gas chromatography-mass spectrometry (GC-MS) to detect and quantify ketones and hydrocarbons (Beller HR, Goh EB, Keasling JD, Appl. Environ. Microbiol. 76: 1212-1223, 2010; Sukovich DJ, Seffernick JL, Richman JE, Gralnick JA, Wackett LP, Appl. Environ. Microbiol. 76: 3850-3862, 2010). While these analyses are powerful and accurate, their labor-intensive nature makes them intractable to high-throughput screening; therefore, methods for rapid identification of bacterial strains that are overproducing hydrocarbons are needed. The use of high-throughput evaluation of bacterial and algal hydrophobic molecule production via Nile red fluorescence from lipids and esters was extended in this study to include hydrocarbons and ketones. This work demonstrated accurate, high-throughput detection of high-level bacterial long-chain ketone and hydrocarbon production by screening for increased fluorescence of the hydrophobic dye Nile red.« less

In vivo spectroscopic photoacoustic tomography imaging of a far red fluorescent protein expressed in the exocrine pancreas of adult zebrafish

NASA Astrophysics Data System (ADS)

Liu, Mengyang; Schmitner, Nicole; Sandrian, Michelle G.; Zabihian, Behrooz; Hermann, Boris; Salvenmoser, Willi; Meyer, Dirk; Drexler, Wolfgang

2014-03-01

Fluorescent proteins brought a revolution in life sciences and biological research in that they make a powerful tool for researchers to study not only the structural and morphological information, but also dynamic and functional information in living cells and organisms. While green fluorescent proteins (GFP) have become a common labeling tool, red-shifted or even near infrared fluorescent proteins are becoming the research focus due to the fact that longer excitation wavelengths are more suitable for deep tissue imaging. In this study, E2-Crimson, a far red fluorescent protein whose excitation wavelength is 611 nm, was genetically expressed in the exocrine pancreas of adult zebrafish. Using spectroscopic all optical detection photoacoustic tomography, we mapped the distribution of E2-Crimson in 3D after imaging the transgenic zebrafish in vivo using two different wavelengths. With complementary morphological information provided by imaging the same fish using a spectral domain optical coherence tomography system, the E2-Crimson distribution acquired from spectroscopic photoacoustic tomography was confirmed in 2D by epifluorescence microscopy and in 3D by histology. To the authors' knowledge, this is the first time a far red fluorescent protein is imaged in vivo by spectroscopic photoacoustic tomography. Due to the regeneration feature of zebrafish pancreas, this work preludes the longitudinal studies of animal models of diseases such as pancreatitis by spectroscopic photoacoustic tomography. Since the effective penetration depth of photoacoustic tomography is beyond the transport mean free path length, other E2-Crimson labeled inner organs will also be able to be studied dynamically using spectroscopic photoacoustic tomography.

Optical Reflectance and Fluorescence for Detecting Nitrogen Needs in Zea mays L.

NASA Technical Reports Server (NTRS)

McMurtrey, J. E.; Middleton, E. M.; Corp. L. A.; Campbell, P. K. Entcheva; Butcher, L. M.; Daughtry, C. S. T.

2003-01-01

Nitrogen (N) status in field grown corn (Zea mays L.) was assessed using spectral techniques. Passive reflectance remote sensing and, both passive and active fluorescence sensing methods were investigated. Reflectance and fluorescence methods are reported to detect changes in the primary plant pigments (chlorophylls a and b; carotenoids) in higher plant species. As a general rule, foliar chlorophyll a (Chl a) and chlorophyll b (Chl b) usually exist in approx.3:l ratio. In plants under stress, Chl b content is affected before Chl a reductions occur. For reflectance, a version of the chlorophyll absorption in reflectance index (CARI) method was tested with narrow bands from the Airborne Imaging Spectroradiometer for Applications (ASIA). CARI minimizes the effects of soil background on the signal from green canopies. A modified CARI (MCARI) was used to track total Chl a levels in the red dip of the spectrum from the corn canopy. A second MCARI was used to track the auxiliary plant pigments (Chl b and the carotenoids) in the yellow/orange/red edge part of the reflectance spectrum. The difference between these two MCARI indices detected variations in N levels across the field plot canopies using ASIA data. At the leaf level, ratios of fluorescence emissions in the blue, green, red and far-red wavelengths sensed responses that were associated with the plant pigments, and were indicative of energy transfer in the photosynthetic process. N stressed corn stands could be distinguish from those with optimally applied N with fluorescence emission spectra obtained from individual corn leaves. Both reflectance and fluorescence methods are sensitive in detecting corn N needs and may be especially powerful in monitoring crop conditions if both types of information can be combined.

RGB-Switchable Porous Electrospun Nanofiber Chemoprobe-Filter Prepared from Multifunctional Copolymers for Versatile Sensing of pH and Heavy Metals.

PubMed

Liang, Fang-Cheng; Kuo, Chi-Ching; Chen, Bo-Yu; Cho, Chia-Jung; Hung, Chih-Chien; Chen, Wen-Chang; Borsali, Redouane

2017-05-17

Novel red-green-blue (RGB) switchable probes based on fluorescent porous electrospun (ES) nanofibers exhibiting high sensitivity to pH and mercury ions (Hg 2+ ) were prepared with one type of copolymer (poly(methyl methacrylatete-co-1,8-naphthalimide derivatives-co-rhodamine derivative); poly(MMA-co-BNPTU-co-RhBAM)) by using a single-capillary spinneret. The MMA, BNPTU, and RhBAM moieties were designed to (i) permit formation of porous fibers, (ii) fluoresce for Hg 2+ detection, and (iii) fluoresce for pH, respectively. The fluorescence emission of BNPTU (fluorescence resonance energy transfer (FRET) donor) changed from green to blue as it detected Hg 2+ . The fluorescence emission of RhBAM (FRET acceptor) was highly selective for pH, changing from nonfluorescent (pH 7) to exhibiting strong red fluorescence (pH 2). The full-color emission of the ES nanofibers included green, red, blue, purple, and white depending on the particular pH and Hg 2+ -concentration combination of the solution. The porous ES nanofibers with 30 nm pores were fabricated using hydrophobic MMA, low-boiling-point solvent, and at a high relative humidity (80%). These porous ES nanofibers had a higher surface-to-volume ratio than did the corresponding thin films, which enhanced their performance. The present study demonstrated that the FRET-based full-color-fluorescence porous nanofibrous membranes, which exhibit on-off switching and can be used as naked eye probes, have potential for application in water purification sensing filters.

Real-time porphyrin detection in plaque and caries: a case study

NASA Astrophysics Data System (ADS)

Timoshchuk, Mari-Alina I.; Ridge, Jeremy S.; Rugg, Amanda L.; Nelson, Leonard Y.; Kim, Amy S.; Seibel, Eric J.

2015-02-01

An ultrathin scanning fiber endoscope, originally developed for cancer diagnosis, was used in a case study to locate plaque and caries. The imaging system incorporated software mitigation of background auto-fluorescence (AF). In conventional fluorescence imaging, varying AF across a tooth surface can mask low-level porphyrin signals. Laser-induced auto-fluorescence signals of dental tissue excited using a 405-nm laser typically produce fluorescence over a wavelength range extending from 440-nm to 750-nm. Anaerobic bacterial metabolism produces various porphyrin species (eg. protoporphyrin IX) that are located in carious enamel, dentin, gingivitis sites, and plaque. In our case study, these porphyrin deposits remained as long as one day after prophylaxis. Imaging the tooth surface using 405-nm excitation and subtracting the natural AF enhances the image contrast of low-level porphyrin deposits, which would otherwise be masked by the high background AF. In a case study, healthy tissues as well as sites of early and advanced caries formations were scanned for visual and quantitative signs of red fluorescence associated with porphyrin species using a background mitigation algorithm. Initial findings show increasing amplitudes of red fluorescence as caries severity increases from early to late stages. Sites of plaque accumulation also displayed red fluorescence similar to that found in carious dental tissue. The use of real-time background mitigation of natural dental AF can enhance the detection of low porphyrin concentrations that are indicators of early stage caries formation.

A pink mouse reports the switch from red to green fluorescence upon Cre-mediated recombination.

PubMed

Hartwich, Heiner; Satheesh, Somisetty V; Nothwang, Hans Gerd

2012-06-14

Targeted genetic modification in the mouse becomes increasingly important in biomedical and basic science. This goal is most often achieved by use of the Cre/loxP system and numerous Cre-driver mouse lines are currently generated. Their initial characterization requires reporter mouse lines to study the in vivo spatiotemporal activity of Cre. Here, we report a dual fluorescence reporter mouse line, which switches expression from the red fluorescent protein mCherry to eGFP after Cre-mediated recombination. Both fluorescent proteins are expressed from the ubiquitously active and strong CAGGS promoter. Among the founders, we noticed a pink mouse line, expressing high levels of the red fluorescent protein mCherry throughout the entire body. Presence of mCherry in the living animal as well as in almost all organs was clearly visible without optical equipment. Upon Cre-activity, mCherry expression was switched to eGFP, demonstrating functionality of this reporter mouse line. The pink mouse presented here is an attractive novel reporter line for fluorescence-based monitoring of Cre-activity. The high expression of mCherry, which is visible to the naked eye, facilitates breeding and crossing, as no genotyping is required to identify mice carrying the reporter allele. The presence of two fluorescent proteins allows in vivo monitoring of recombined and non-recombined cells. Finally, the pink mouse is an eye-catching animal model to demonstrate the power of transgenic techniques in teaching courses.

ICT-Isomerization-Induced Turn-On Fluorescence Probe with a Large Emission Shift for Mercury Ion: Application in Combinational Molecular Logic.

PubMed

Bhatta, Sushil Ranjan; Mondal, Bijan; Vijaykumar, Gonela; Thakur, Arunabha

2017-10-02

A unique turn-on fluorescent device based on a ferrocene-aminonaphtholate derivative specific for Hg 2+ cation was developed. Upon binding with Hg 2+ ion, the probe shows a dramatic fluorescence enhancement (the fluorescence quantum yield increases 58-fold) along with a large red shift of 68 nm in the emission spectrum. The fluorescence enhancement with a red shift may be ascribed to the combinational effect of C═N isomerization and an extended intramolecular charge transfer (ICT) mechanism. The response was instantaneous with a detection limit of 2.7 × 10 -9 M. Upon Hg 2+ recognition, the ferrocene/ferrocenium redox peak was anodically shifted by ΔE 1/2 = 72 mV along with a "naked eye" color change from faint yellow to pale orange for this metal cation. Further, upon protonation of the imine nitrogen, the present probe displays a high fluorescence output due to suppression of the C═N isomerization process. Upon deprotonation using strong base, the fluorescence steadily decreases, which indicates that H + and OH - can be used to regulate the off-on-off fluorescence switching of the present probe. Density functional theory studies revealed that the addition of acid leads to protonation of the imine N (according to natural bond orbital analysis), and the resulting iminium proton forms a strong H-bond (2.307 Å) with one of the triazole N atoms to form a five-membered ring, which makes the molecule rigid; hence, enhancement of the ICT process takes place, thereby leading to a fluorescence enhancement with a red shift. The unprecedented combination of H + , OH - , and Hg 2+ ions has been used to generate a molecular system exhibiting the INHIBIT-OR combinational logic operation.

Proximity-Induced Covalent Labeling of Proteins with a Reactive Fluorophore-Binding Peptide Tag.

PubMed

Sunbul, Murat; Nacheva, Lora; Jäschke, Andres

2015-08-19

Labeling of proteins with fluorescent dyes in live cells enables the investigation of their roles in biological systems by fluorescence microscopy. Because the labeling procedure should not disturb the native function of the protein of interest, it is of high importance to find the optimum labeling method for the problem to be studied. Here, we developed a rapid one-step method to covalently and site-specifically label proteins with a TexasRed fluorophore in vitro and in live bacteria. To this end, a genetically encodable TexasRed fluorophore-binding peptide (TR512) was converted into a reactive tag (ReacTR) by adjoining a cysteine residue which rapidly reacts with N-α-chloroacetamide-conjugated TexasRed fluorophore owing to the proximity effect; ReacTR tag first binds to the TexasRed fluorophore and this interaction brings the nucleophilic cysteine and the electrophilic N-α-chloroacetamide groups in close proximity. Our method has several advantages over existing methods: (i) it utilizes a peptide tag much smaller than fluorescent proteins, the SNAP, CLIP, or HaLo tags; (ii) it allows for labeling of proteins with a small, photostable, red-emitting TexasRed fluorophore; (iii) the probe used is very easy to synthesize; (iv) no enzyme is required to transfer the fluorophore to the peptide tag; and (v) labeling yields a stable covalent product in a very fast reaction.

Structural characterization of acylimine-containing blue and red chromophores in mTagBFP and TagRFP fluorescent proteins.

PubMed

Subach, Oksana M; Malashkevich, Vladimir N; Zencheck, Wendy D; Morozova, Kateryna S; Piatkevich, Kiryl D; Almo, Steven C; Verkhusha, Vladislav V

2010-04-23

We determined the 2.2 A crystal structures of the red fluorescent protein TagRFP and its derivative, the blue fluorescent protein mTagBFP. The crystallographic analysis is consistent with a model in which TagRFP has the trans coplanar anionic chromophore with the conjugated pi-electron system, similar to that of DsRed-like chromophores. Refined conformation of mTagBFP suggests the presence of an N-acylimine functionality in its chromophore and single C(alpha)-C(beta) bond in the Tyr64 side chain. Mass spectrum of mTagBFP chromophore-bearing peptide indicates a loss of 20 Da upon maturation, whereas tandem mass spectrometry reveals that the C(alpha)-N bond in Leu63 is oxidized. These data indicate that mTagBFP has a new type of the chromophore, N-[(5-hydroxy-1H-imidazole-2-yl)methylidene]acetamide. We propose a chemical mechanism in which the DsRed-like chromophore is formed via the mTagBFP-like blue intermediate. (c) 2010 Elsevier Ltd. All rights reserved.

Extended Stokes shift in fluorescent proteins: chromophore-protein interactions in a near-infrared TagRFP675 variant.

PubMed

Piatkevich, Kiryl D; Malashkevich, Vladimir N; Morozova, Kateryna S; Nemkovich, Nicolai A; Almo, Steven C; Verkhusha, Vladislav V

2013-01-01

Most GFP-like fluorescent proteins exhibit small Stokes shifts (10-45 nm) due to rigidity of the chromophore environment that excludes non-fluorescent relaxation to a ground state. An unusual near-infrared derivative of the red fluorescent protein mKate, named TagRFP675, exhibits the Stokes shift, which is 30 nm extended comparing to that of the parental protein. In physiological conditions, TagRFP675 absorbs at 598 nm and emits at 675 nm that makes it the most red-shifted protein of the GFP-like protein family. In addition, its emission maximum strongly depends on the excitation wavelength. Structures of TagRFP675 revealed the common DsRed-like chromophore, which, however, interacts with the protein matrix via an extensive network of hydrogen bonds capable of large flexibility. Based on the spectroscopic, biochemical, and structural analysis we suggest that the rearrangement of the hydrogen bond interactions between the chromophore and the protein matrix is responsible for the TagRFP675 spectral properties.

Fluorescent Protein-Based Quantification of Alternative Splicing of a Target Cassette Exon in Mammalian Cells.

PubMed

Gurskaya, N G; Staroverov, D B; Lukyanov, K A

2016-01-01

Alternative splicing is an important mechanism of regulation of gene expression and expansion of proteome complexity. Recently we developed a new fluorescence reporter for quantitative analysis of alternative splicing of a target cassette exon in live cells (Gurskaya et al., 2012). It consists of a specially designed minigene encoding red and green fluorescent proteins (Katushka and TagGFP2) and a fragment of the target gene between them. Skipping or inclusion of the alternative exon induces a frameshift; ie, alternative exon length must not be a multiple of 3. Finally, red and green fluorescence intensities of cells expressing this reporter are used to estimate the percentage of alternative (exon-skipped) and normal (exon-retained) transcripts. Here, we provide a detailed description of design and application of the fluorescence reporter of a target alternative exon splicing in mammalian cell lines. © 2016 Elsevier Inc. All rights reserved.

Red fluorescent chitosan nanoparticles grafted with poly(2-methacryloyloxyethyl phosphorylcholine) for live cell imaging.

PubMed

Wang, Ke; Fan, Xingliang; Zhang, Xiaoyong; Zhang, Xiqi; Chen, Yi; Wei, Yen

2016-08-01

Poly(2-methacryloyloxyethyl phosphorylcholine) conjugated red fluorescent chitosan nanoparticles (GCC-pMPC) were facilely fabricated by "grafting from" method via surface initiated atom transfer radical polymerization (ATRP). Firstly, glutaraldehyde crosslinked red fluorescent chitosan nanoparticles (GCC NPs) with many amino groups and hydroxyl groups on their surface were prepared, which were then reacted with 2-bromoisobutyryl bromide to form GCC-Br; subsequently, poly(MPC) (pMPC) brushes were grafted onto GCC NPs surface using GCC-Br as initiator via ATRP. Compared with PEGylated nanoparticles, zwitterionic polymers modified nanoparticles demonstrated better performance in their cellular uptake. Moreover, the obtained GCC-pMPC demonstrated excellent water-dispersibility, biocompatibility, and photostability, which made them highly potential for long-term tracing applications. Importantly, the successful live cell imaging of GCC-pMPC would remarkably advance the research of their further bioapplications. Copyright © 2016 Elsevier B.V. All rights reserved.

Synthesis and characterization of a new fluorogenic substrate for alpha-galactosidase

PubMed Central

Shi, Zhen-Dan; Motabar, Omid; Goldin, Ehud; Liu, Ke; Southall, Noel; Sidransky, Ellen; Austin, Christopher P.; Griffiths, Gary L.

2009-01-01

Alpha-galactosidase A hydrolyzes the terminal alpha-galactosyl moieties from glycolipids and glycoproteins in lysosomes. Mutations in α-galactosidase cause lysosomal accumulation of the glycosphingolipid, globotriaosylceramide, which leads to Fabry disease. Small-molecule chaperones that bind to mutant enzyme proteins and correct their misfolding and mistrafficking have emerged as a potential therapy for Fabry disease. We have synthesized a red fluorogenic substrate, resorufinyl α-D-galactopyranoside, for a new α-galactosidase enzyme assay. This assay can be measured continuously at lower pH values, without the addition of a stop solution, due to the relatively low pKa of resorufin (~6). In addition, the assay emits red fluorescence, which can significantly reduce interferences due to compound fluorescence and dust/lint as compared to blue fluorescence. Therefore, this new red fluorogenic substrate and the resulting enzyme assay can be used in high-throughput screening to identify small-molecule chaperones for Fabry disease. PMID:19521690

A Zn-porphyrin complex contributes to bright red color in Parma ham.

PubMed

Wakamatsu, J; Nishimura, T; Hattori, A

2004-05-01

The Italian traditional dry-cured ham (Parma ham) shows a stable bright red color that is achieved without the use of nitrite and/or nitrate. In this study we examined the pigment spectroscopically, fluoroscopically and by using HPLC and ESI-HR-MASS analysis. Porphyrin derivative other than acid hematin were contained in the HCl-containing acetone extract from Parma ham. A strong fluorescence peak at 588 nm and a weak fluorescence peak at 641 nm were observed. By HPLC analysis the acetone extract of Parma ham was observed at the single peak, which eluted at the same time as Zn-protoporphyrin IX and emitted fluorescence. The results of ESI-HR-MS analysis showed both agreement with the molecular weight of Zn-protoporphyrin IX and the characteristic isotope pattern caused by Zn isotopes. These results suggest that the bright red color in Parma ham is caused by Zn-protoporphyrin IX.

Mechanisms of Formation and Structure of Chromophores of Fluorescent Proteins from Anthoza Species

DTIC Science & Technology

2005-03-01

green fluorescent protein (GFP) from the jellyfish Aequorea victoria ...investigate chemical structures and mechanisms of formation of chromophores within proteins of Green Fluorescent Protein (GFP) family. The project... structure . In this part of work we have shown that a fluorescent protein from Dendronephthya sp. transforms from the green - to the red-emitting

The potential of a fluorescent-based approach for bioassay of antifungal agents against chili anthracnose disease in Thailand.

PubMed

Chutrakul, Chanikul; Khaokhajorn, Pratoomporn; Auncharoen, Patchanee; Boonruengprapa, Tanapong; Mongkolporn, Orarat

2013-01-01

Severe chili anthracnose disease in Thailand is caused by Colletotrichum gloeosporioides and C. capsici. To discover anti-anthracnose substances we developed an efficient dual-fluorescent labeling bioassay based on a microdilution approach. Indicator strains used in the assay were constructed by integrating synthetic green fluorescent protein (sGFP) and Discosoma sp. red fluorescent protein (DsRedExp) genes into the genomes of C. gloeosporioides or C. capsici respectively. Survival of co-spore cultures in the presence of inhibitors was determined by the expression levels of these fluorescent proteins. This developed assay has high potential for utilization in the investigation of selective inhibition activity to either one of the pathogens as well as the broad-range inhibitory effect against both pathogens. The value of using the dual-fluorescent assay is rapid, reliable, and consistent identification of anti-anthracnose agents. Most of all, the assay enables the identification of specific inhibitors under the co-cultivation condition.

Porphyrin involvement in redshift fluorescence in dentin decay

NASA Astrophysics Data System (ADS)

Slimani, A.; Panayotov, I.; Levallois, B.; Cloitre, T.; Gergely, C.; Bec, N.; Larroque, C.; Tassery, H.; Cuisinier, F.

2014-05-01

The aim of this study was to evaluate the porphyrin involvement in the red fluorescence observed in dental caries with Soprolife® light-induced fluorescence camera in treatments mode (SOPRO, ACTEON Group, La Ciotat, France) and Vistacam® camera (DÜRR DENTAL AG, Bietigheim-Bissingen, Germany). The International Caries Detection and Assessment System (ICDAS) was used to rand the samples. Human teeth cross-sections, ranked from ICDAS score 0 to 6, were examined by epi-fluorescence microscopy and Confocal Raman microscopy. Comparable studies were done with Protoporphyrin IX, Porphyrin I and Pentosidine solutions. An RGB analysis of Soprolife® images was performed using ImageJ Software (1.46r, National Institutes of Health, USA). Fluorescence spectroscopy and MicroRaman spectroscopy revealed the presence of Protoporphyrin IX, in carious enamel, dentin and dental plaque. However, the presence of porphyrin I and pentosidine cannot be excluded. The results indicated that not only porphyrin were implicated in the red fluorescence, Advanced Glygation Endproducts (AGEs) of the Maillard reaction also contributed to this phenomenon.

8-Anilino-1-naphthalenesulfonate/Layered Double Hydroxide Ultrathin Films: Small Anion Assembly and Its Potential Application as a Fluorescent Biosensor.

PubMed

Zhang, Ping; Li, Ling; Zhao, Yun; Tian, Zeyun; Qin, Yumei; Lu, Jun

2016-09-06

The fluorescent dye 8-anilino-1-naphthalenesulfonate (ANS) is a widely used fluorescent probe molecule for biochemistry analysis. This paper reported the fabrication of ANS/layered double hydroxide nanosheets (ANS/LDH)n ultrathin films (UTFs) via the layer-by-layer small anion assembly technique based on electrostatic interaction and two possible weak interactions: hydrogen-bond and induced electrostatic interactions between ANS and positive-charged LDH nanosheets. The obtained UTFs show a long-range-ordered periodic layered stacking structure and weak fluorescence in dry air or water, but it split into three narrow strong peaks in a weak polarity environment induced by the two-dimensional (2D) confinement effect of the LDH laminate; the fluorescence intensity increases with decreasing the solvent polarity, concomitant with the blue shift of the emission peaks, which show good sensoring reversibility. Meanwhile, the UTFs exhibit selective fluorescence enhancement to the bovine serum albumin (BSA)-like protein biomolecules, and the rate of fluorescence enhancement with the protein concentration is significantly different with the different protein aggregate states. The (ANS/LDH)n UTF has the potential to be a novel type of biological flourescence sensor material.

A fluorogenic probe for SNAP-tagged plasma membrane proteins based on the solvatochromic molecule Nile Red.

PubMed

Prifti, Efthymia; Reymond, Luc; Umebayashi, Miwa; Hovius, Ruud; Riezman, Howard; Johnsson, Kai

2014-03-21

A fluorogenic probe for plasma membrane proteins based on the dye Nile Red and SNAP-tag is introduced. It takes advantage of Nile Red, a solvatochromic molecule highly fluorescent in an apolar environment, such as cellular membranes, but almost dark in a polar aqueous environment. The probe possesses a tuned affinity for membranes allowing its Nile Red moiety to insert into the lipid bilayer of the plasma membrane, becoming fluorescent, only after its conjugation to a SNAP-tagged plasma membrane protein. The fluorogenic character of the probe was demonstrated for different SNAP-tag fusion proteins, including the human insulin receptor. This work introduces a new approach for generating a powerful turn-on probe for "no-wash" labeling of plasma membrane proteins with numerous applications in bioimaging.

Nile Red Detection of Bacterial Hydrocarbons and Ketones in a High-Throughput Format

PubMed Central

Pinzon, Neissa M.; Aukema, Kelly G.; Gralnick, Jeffrey A.; Wackett, Lawrence P.

2011-01-01

ABSTRACT A method for use in high-throughput screening of bacteria for the production of long-chain hydrocarbons and ketones by monitoring fluorescent light emission in the presence of Nile red is described. Nile red has previously been used to screen for polyhydroxybutyrate (PHB) and fatty acid esters, but this is the first report of screening for recombinant bacteria making hydrocarbons or ketones. The microtiter plate assay was evaluated using wild-type and recombinant strains of Shewanella oneidensis and Escherichia coli expressing the enzyme OleA, previously shown to initiate hydrocarbon biosynthesis. The strains expressing exogenous Stenotrophomonas maltophilia oleA, with increased levels of ketone production as determined by gas chromatography-mass spectrometry, were distinguished with Nile red fluorescence. Confocal microscopy images of S. oneidensis oleA-expressing strains stained with Nile red were consistent with a membrane localization of the ketones. This differed from Nile red staining of bacterial PHB or algal lipid droplets that showed intracellular inclusion bodies. These results demonstrated the applicability of Nile red in a high-throughput technique for the detection of bacterial hydrocarbons and ketones. PMID:21712420

Method for flow cytometric monitoring of Renibacterium salmoninarum inactivation

USGS Publications Warehouse

Pascho, R.J.; Ongerth, J.E.

2000-01-01

The slow growth of Renibacterium salmoninarum limits the usefulness of culture as a research tool. Development of a 2-color flow cytometric assay to quantify the proportions of live and dead R. salmoninarum in a test population is described. Bacteria were simultaneously stained with fluorescein isothiocyanate-conjugated immunoglobulin and exposed to the exclusion dye propidium iodide. Propidium iodide red fluorescence profiles of control groups of untreated and killed R. salmoninarum were compared with those for bacteria exposed to chlorine. Bacterial inactivation was based on mean red fluorescence intensity, and analyzed by high-red fluorescence intensity (HRFI) and curve subtraction (CS) analyses. When the concentration of R. salmoninarum was 8.65 x 106 bacteria ml-1 and the bacteria exposed to chlorine at 1 mg l-1 for periods from 1 to 20 min (high-Rs assessment), the mean red fluorescence intensity of the profile for each chlorine-exposure group was higher than that for the untreated control (p < 0.0001). When the concentration of R. salmoninarum was reduced to 1.76 x 106 bacteria ml-1 and exposed to 0.8 mg l-1 free chlorine level for periods from 20 s to 5 min (reduced-Rs assessment), the mean red fluorescence intensities of the exposure groups were higher than that for the untreated control only when the R. salmoninarum was exposed to chlorine for at least 1 min (p ??? 0.01). On the basis of red fluorescence intensity, the proportion of dead cells generally increased with the duration of chlorine exposure. Whereas the rates of inactivation derived from the HRFI and CS analyses did not correlate with the duration of exposure in the high-Rs assessment (r2 ??? 0.27), there was a correlation between these estimates and the duration of exposure in the reduced-Rs assessment (r2 ??? 0.92). Because of the rapid loss of culturable R. salmoninarum in both assessments following chlorine exposure, neither the duration of exposure nor the inactivation estimates correlated with bacteriological culture (r2 ??? 0.22). In both assessments, there was a correlation between the estimates of inactivation based upon HRFI and CS analyses (r2 > 0.99). These results suggest that flow cytometry can be used as a supplementary or alternative method to bacteriological culture for monitoring the inactivation of R. salmoninarum.

Extended Fluorescent Resonant Energy Transfer in DNA Constructs

NASA Astrophysics Data System (ADS)

Oh, Taeseok

This study investigates the use of surfactants and metal cations for the enhancement of long range fluorescent resonant energy transfer (FRET) and the antenna effect in DNA structures with multiple fluorescent dyes. Double-stranded (ds) DNA structures were formed by hybridization of 21mer DNA oligonucleotides with different arrangements of three fluorescent TAMRA donor dyes with two different complementary 21mer oligonucleotides with one fluorescent TexasRed acceptor dye. In such DNA structures, hydrophobic interactions between the fluorescent dyes in close proximity produces dimerization which along with other quenching mechanisms leads to significant reduction of fluorescent emission properties. Addition of the surfactants Triton X-100, cetyltrimethyl ammonium bromide (CTAB) and sodium dodecyl sulfate (SDS) along with sodium cations (Na+) and divalent magnesium cations (Mg 2+) were tested for their ability to reduce quenching of the fluorescent dyes and improve overall fluorescent emission, the long range FRET and the antenna effect properties. When the neutral (uncharged) surfactant Triton X-100 was added to the FRET ds-DNA hybrid structures with three TAMRA donors and one TexasRed acceptor, dye dimerization and emission quenching remained unaffected. However, for the positively charged CTAB surfactant at concentrations of 100 uM or higher, the neutralization of the negatively charged ds-DNA backbone by the cationic surfactant micelles was found to reduce TAMRA dye dimerization and emission quenching and improve TexasRed quantum yield, resulting in much higher FRET efficiencies and an enhanced antenna effect. This improvement is likely due to the CTAB molecules covering or sheathing the fluorescent donor and acceptor dyes which breaks up the dimerized dye complexes and prevents further quenching from interactions with water molecules and guanine bases in the DNA structure. While the negatively charged SDS surfactant alone was not able to reduce dimerization and emission quenching due to repulsive forces between DNA and SDS micelles, the addition of cations such as sodium ions (Na+) and divalent magnesium ions (Mg2+) did lead to a significant reduction in the dimerization and emission quenching resulting in much higher FRET efficiency and an enhanced antenna effect. It appears that when the repulsive electrostatic forces are screened by the cations (Mg2+ in particular), the SDS micelles can approach the FRET ds-DNA structures thereby sheathing or insulating the TAMRA and TexasRed dyes. Overall, the study provides a viable strategy for using combinations of surfactants and cations to reduce adverse fluorescent dye and other quenching mechanisms and improve the overall long distance FRET efficiency and the antenna effect in DNA structures with multi-donor and single acceptor fluorescent dye groups.

A broad-host range dual-fluorescence reporter system for gene expression analysis in Gram-negative bacteria.

PubMed

Hennessy, Rosanna C; Christiansen, Line; Olsson, Stefan; Stougaard, Peter

2018-01-01

Fluorescence-based reporter systems are valuable tools for studying gene expression dynamics in living cells. Here we describe a dual-fluorescence reporter system carrying the red fluorescent marker mCherry and the blue fluorescent protein EBFP2 enabling the simultaneous analysis of two promoters in broad-host range autofluorescent Gram-negative bacteria. Copyright © 2017 Elsevier B.V. All rights reserved.

Hydrophobic-Sheath Segregated Macromolecular Fluorophores: Colloidal Nanoparticles of Polycaprolactone-Grafted Conjugated Polymers with Bright Far-Red/Near-Infrared Emission for Biological Imaging.

PubMed

Yang, Cangjie; Liu, Hui; Zhang, Yingdan; Xu, Zhigang; Wang, Xiaochen; Cao, Bin; Wang, Mingfeng

2016-05-09

This article describes molecular design, synthesis and characterization of colloidal nanoparticles containing polycaprolactone-grafted conjugated polymers that exhibit strong far red/near-infrared (FR/NIR) fluorescence for bioimaging. Specifically, we synthesized two kinds of conjugated polymer bottle brushes (PFTB(out)-g-PCL and PFTB(in)-g-PCL) with different positions of the hexyl groups on the thiophene rings. A synthetic amphiphilic block copolymer PCL-b-POEGMA was employed as surfactants to encapsulate PFTB-g-PCL polymers into colloidal nanoparticles (denoted as "nanoREDs") in aqueous media. The chain length of the PCL side chains in PFTB-g-PCL played a critical role in determining the fluorescence properties in both bulk solid states and the colloidal nanoparticles. Compared to semiconducting polymer dots (Pdots) composed of PFTB(out) without grafted PCL, nanoRED(out) showed at least four times higher fluorescence quantum yield (∼20%) and a broader emission band centered at 635 nm. We further demonstrated the application of this new class of nanoREDs for effective labeling of L929 cells and HeLa cancer cells with good biocompatibility. This strategy of hydrophobic-sheath segregated macromolecular fluorophores is expected to be applicable to a broad range of conjugated polymers with tunable optical properties for applications such as bioimaging.

A Photostable Silicon Rhodamine Platform for Optical Voltage Sensing

PubMed Central

Huang, Yi-Lin; Walker, Alison S.; Miller, Evan W.

2015-01-01

This paper describes the design and synthesis of a photostable, far-red to near-infrared (NIR) platform for optical voltage sensing. We developed a new, sulfonated silicon rhodamine fluorophore and integrated it with a phenylenevinylene molecular wire to create a Berkeley Red Sensor of Transmembrane potential, or BeRST 1 (“burst”). BeRST 1 is the first member of a class of farred to NIR voltage sensitive dyes that make use of a photoinduced electron transfer (PeT) trigger for optical interrogation of membrane voltage. We show that BeRST 1 displays bright, membrane-localized fluorescence in living cells, high photostability, and excellent voltage sensitivity in neurons. Depolarization of the plasma membrane results in rapid fluorescence increases (24% ΔF/F per 100 mV). BeRST 1 can be used in conjunction with fluorescent stains for organelles, Ca2+ indicators, and voltage-sensitive fluorescent proteins. In addition, the red-shifted spectral profile of BeRST 1, relative to commonly employed optogenetic actuators like ChannelRhodopsin2 (ChR2), which require blue light, enables optical electrophysiology in neurons. The high speed, sensitivity, photostability and long-wavelength fluorescence profiles of BeRST 1 make it a useful platform for the non-invasive, optical dissection of neuronal activity. PMID:26237573

Fluorescence lifetime imaging of lipids during 3T3-L1 cell differentiation

NASA Astrophysics Data System (ADS)

Song, Young Sik; Won, Young Jae; Lee, Sang-Hak; Kim, Dug Young

2014-03-01

Obesity is becoming a big health problem in these days. Since increased body weight is due to increased number and size of the triglyceride-storing adipocytes, many researchers are working on differentiation conditions and processes of adipocytes. Adipocytes also work as regulators of whole-body energy homeostasis by secreting several proteins that regulate processes as diverse as haemostasis, blood pressure, immune function, angiogenesis and energy balance. 3T3-L1 cells are widely used cell line for studying adipogenesis because it can differentiate into an adipocyte-like phenotype under appropriate conditions. In this paper, we propose an effective fluorescence lifetime imaging technique which can easily distinguish lipids in membrane and those in lipid droplets. Nile red dyes are attached to lipids in 3T3-L1 cells. Fluorescence lifetime images were taken for 2 week during differentiation procedure of 3T3-L1 cells into adipocytes. We used 488 nm pulsed laser with 5MHz repetition rate and emission wavelength is 520 nm of Nile Red fluorescent dye. Results clearly show that the lifetime of Nile red in lipid droplets are smaller than those in cell membrane. Our results suggest that fluorescence lifetime imaging can be a very powerful tool to monitor lipid droplet formation in adipocytes from 3T3-L1 cells.

A Biocompatible and Biodegradable Protein Hydrogel with Green and Red Autofluorescence: Preparation, Characterization and In Vivo Biodegradation Tracking and Modeling

PubMed Central

Ma, Xiaoyu; Sun, Xiangcheng; Hargrove, Derek; Chen, Jun; Song, Donghui; Dong, Qiuchen; Lu, Xiuling; Fan, Tai-Hsi; Fu, Youjun; Lei, Yu

2016-01-01

Because of its good biocompatibility and biodegradability, albumins such as bovine serum albumin (BSA) and human serum albumin (HSA) have found a wide range of biomedical applications. Herein, we report that glutaraldehyde cross-linked BSA (or HSA) forms a novel fluorescent biological hydrogel, exhibiting new green and red autofluorescence in vitro and in vivo without the use of any additional fluorescent labels. UV-vis spectra studies, in conjunction with the fluorescence spectra studies including emission, excitation and synchronous scans, indicated that three classes of fluorescent compounds are presumably formed during the gelation process. SEM, FTIR and mechanical tests were further employed to investigate the morphology, the specific chemical structures and the mechanical strength of the as-prepared autofluorescent hydrogel, respectively. Its biocompatibility and biodegradability were also demonstrated through extensive in vitro and in vivo studies. More interestingly, the strong red autofluorescence of the as-prepared hydrogel allows for conveniently and non-invasively tracking and modeling its in vivo degradation based on the time-dependent fluorescent images of mice. A mathematical model was proposed and was in good agreement with the experimental results. The developed facile strategy to prepare novel biocompatible and biodegradable autofluorescent protein hydrogels could significantly expand the scope of protein hydrogels in biomedical applications. PMID:26813916

Syphilis Test

MedlinePlus

... Time and International Normalized Ratio (PT/INR) PSEN1 Quantitative Immunoglobulins Red Blood Cell (RBC) Antibody Identification Red ... Sources Ask Us Also Known As Venereal Disease Research Laboratory VDRL Rapid Plasma Reagin RPR Fluorescent Treponemal ...

Inner filter effect and the onset of concentration dependent red shift of synchronous fluorescence spectra.

PubMed

Tarai, Madhumita; Mishra, Ashok Kumar

2016-10-12

The phenomenon of concentration dependent red shift, often observed in synchronous fluorescence spectra (SFS) of monofluorophoric as well as multifluorophoric systems at high chromophore concentrations, is known to have good analytical advantages. This was previously understood in terms of large inner filter effect (IFE) through the introduction of a derived absorption spectral profile that closely corresponds to the SFS profile. Using representative monofluorophoric and multifluorophoric systems, it is now explained how the SF spectral maximum changes with concentration of the fluorophore. For dilute solutions of monofluorophores the maximum is unchanged as expected. It is shown here that the onset of red shift of SFS maximum of both the mono as well as the multifluorophoric systems must occur at the derived absorption spectral parameter value of 0.32 that corresponds to the absorbance value of 0.87. This value is unique irrespective of the nature of the fluorophore under study. For monofluorophoric systems, the wavelength of derived absorption spectral maximum and the wavelength of synchronous fluorescence spectral maximum closely correspond with each other in the entire concentration range. In contrast, for multifluorophoric systems like diesel and aqueous humic acid, large deviations were noted that could be explained as to be due to the presence of non-fluorescing chromophores in the system. This work bridges the entire fluorophore concentration range over which the red shift of SFS maximum sets in; and in the process it establishes the importance of the derived absorption spectral parameter in understanding the phenomenon of concentration dependent red shift of SFS maximum. Copyright © 2016 Elsevier B.V. All rights reserved.

Seed-to-seed growth of superdwarf wheat and arabidopsis using red light-emitting diodes (LED's): A report on baseline tests conducted for NASA's proposed Plant Research Unit (PRU)

NASA Technical Reports Server (NTRS)

Goins, G. D.; Yorio, N. C.; Sanwo, M. M.; Brown, C. S.

1996-01-01

To determine the influence of narrow-spectrum red light-emitting diodes (LED's) on plant growth and seed production, wheat (Triticum aestivum L.cv Superdwarf) and Arabidopsis (Arabidopsis thaliana (L.) Heynh, race Columbia) plants were grown under red LED's (peak emission 660 nm) and compared to plants grown under daylight fluorescent (white) light and red LED's supplemented with either 1 percent or 10 percent blue fluorescent (BF) light. Wheat growth under red LED's alone appeared normal, whereas Arabidopsis under red LED's alone developed curled leaf margins and a spiraling growth pattern. Both wheat and Arabidopsis under red LED's alone or red LED's + 1 percent BF light had significantly lower seed yield than plants grown under white light. However, the addition of 10 percent BF light to red LED's partially alleviated the adverse effect of red LED's on yield. Irrespective of the light treatment, viable seeds were produced by wheat(75-92 percent germination rate) and Arabidopsis (85-100 percent germination rate). These results indicate that wheat, and to a lesser extent Arabidopsis, can be successfully grown under red LED's alone, but supplemental blue light is required with red LED's to sufficiently match the growth characteristics and seed yield associated with plants grown under white light.

Demonstration of Smart Fluorescent and Self-Healing Coatings for Severely Corrosive Environments at Vehicle Wash Facilities

DTIC Science & Technology

2009-08-01

as well as pipe and tank exteriors providing early detection of coating erosion, cracks , and intercoat blistering. A fluorescing coating used ERDC...poor with widespread areas of peeling and cracking on the exterior siding. Areas of exposed galvanizing were rusting. Structural steel elements...and application of TT-P-86 Type 2 red lead paint and red and white colored alkyd enamel topcoats. The average dry film thickness on the exterior

Spatially controlled fabrication of a bright fluorescent nanodiamond-array with enhanced far-red Si-V luminescence.

PubMed

Singh, Sonal; Thomas, Vinoy; Martyshkin, Dmitry; Kozlovskaya, Veronika; Kharlampieva, Eugenia; Catledge, Shane A

2014-01-31

We demonstrate a novel approach to precisely pattern fluorescent nanodiamond-arrays with enhanced far-red intense photostable luminescence from silicon-vacancy (Si-V) defect centers. The precision-patterned pre-growth seeding of nanodiamonds is achieved by a scanning probe 'dip-pen' nanolithography technique using electrostatically driven transfer of nanodiamonds from 'inked' cantilevers to a UV-treated hydrophilic SiO2 substrate. The enhanced emission from nanodiamond dots in the far-red is achieved by incorporating Si-V defect centers in a subsequent chemical vapor deposition treatment. The development of a suitable nanodiamond ink and mechanism of ink transport, and the effect of humidity and dwell time on nanodiamond patterning are investigated. The precision patterning of as-printed (pre-CVD) arrays with dot diameter and dot height as small as 735 nm ± 27 nm and 61 nm ± 3 nm, respectively, and CVD-treated fluorescent ND-arrays with consistently patterned dots having diameter and height as small as 820 nm ± 20 nm and, 245 nm ± 23 nm, respectively, using 1 s dwell time and 30% RH is successfully achieved. We anticipate that the far-red intense photostable luminescence (~738 nm) observed from Si-V defect centers integrated in spatially arranged nanodiamonds could be beneficial for the development of next generation fluorescence-based devices and applications.

Fluorescence probe techniques to monitor protein adsorption-induced conformation changes on biodegradable polymers.

PubMed

Benesch, Johan; Hungerford, Graham; Suhling, Klaus; Tregidgo, Carolyn; Mano, João F; Reis, Rui L

2007-08-15

The study of protein adsorption and any associated conformational changes on interaction with biomaterials is of great importance in the area of implants and tissue constructs. This study aimed to evaluate some fluorescent techniques to probe protein conformation on a selection of biodegradable polymers currently under investigation for biomedical applications. Because of the fluorescence emanating from the polymers, the use of monitoring intrinsic protein fluorescence was precluded. A highly solvatochromic fluorescent dye, Nile red, and a well-known protein label, fluorescein isothiocyanate, were employed to study the adsorption of serum albumin to polycaprolactone and to some extent also to two starch-containing polymer blends (SPCL and SEVA-C). A variety of fluorescence techniques, steady state, time resolved, and imaging were employed. Nile red was found to leach from the protein, while fluorescein isothiocyanate proved useful in elucidating a conformational change in the protein and the observation of protein aggregates adsorbed to the polymer surface. These effects were seen by making use of the phenomenon of energy migration between the fluorescent tags to monitor interprobe distance and the use of fluorescence lifetime imaging to ascertain the surface packing of the protein on polymer.

Discrimination of corn from monocotyledonous weeds with ultraviolet (UV) induced fluorescence.

PubMed

Panneton, Bernard; Guillaume, Serge; Samson, Guy; Roger, Jean-Michel

2011-01-01

In production agriculture, savings in herbicides can be achieved if weeds can be discriminated from crop, allowing the targeting of weed control to weed-infested areas only. Previous studies demonstrated the potential of ultraviolet (UV) induced fluorescence to discriminate corn from weeds and recently, robust models have been obtained for the discrimination between monocots (including corn) and dicots. Here, we developed a new approach to achieve robust discrimination of monocot weeds from corn. To this end, four corn hybrids (Elite 60T05, Monsanto DKC 26-78, Pioneer 39Y85 (RR), and Syngenta N2555 (Bt, LL)) and four monocot weeds (Digitaria ischaemum (Schreb.) I, Echinochloa crus-galli (L.) Beauv., Panicum capillare (L.), and Setaria glauca (L.) Beauv.) were grown either in a greenhouse or in a growth cabinet and UV (327 nm) induced fluorescence spectra (400 to 755 nm) were measured under controlled or uncontrolled ambient light intensity and temperature. This resulted in three contrasting data sets suitable for testing the robustness of discrimination models. In the blue-green region (400 to 550 nm), the shape of the spectra did not contain any useful information for discrimination. Therefore, the integral of the blue-green region (415 to 455 nm) was used as a normalizing factor for the red fluorescence intensity (670 to 755 nm). The shape of the normalized red fluorescence spectra did not contribute to the discrimination and in the end, only the integral of the normalized red fluorescence intensity was left as a single discriminant variable. Applying a threshold on this variable minimizing the classification error resulted in calibration errors ranging from 14.2% to 15.8%, but this threshold varied largely between data sets. Therefore, to achieve robustness, a model calibration scheme was developed based on the collection of a calibration data set from 75 corn plants. From this set, a new threshold can be estimated as the 85% quantile on the cumulative frequency curve of the integral of the normalized red fluorescence. With this approach the classification error was nearly constant (16.0% to 18.5%), thereby indicating the potential of UV-induced fluorescence to reliably discriminate corn from monocot weeds.

Filling-In of Broad Far-Red Solar Lines by Terrestrial Fluorescence and Atmospheric Raman Scattering as Detected by SCIAMACHY Satellite Measurements

NASA Technical Reports Server (NTRS)

Joiner, J.; Yoshida, Y.; Vasilkov, A. P.; Middleton, E. M.; Campbell, P. K. E.; Yoshida, Y.; Kuze, A.; Corp, L. A.

2011-01-01

Global mapping of terrestrial vegetation fluorescence from space has recently been accomplished with high spectral resolution measurements from the Japanese Greenhouse gases Observing SATellite (GOSAT). These data are of interest because they can potentially provide global information on the functional status of vegetation including light use efficiency and global primary productivity that can be used for global carbon cycle modeling. Quantifying the impact of fluorescence on the O2-A band is important as this band is used for cloud- and aerosol-characterization for other trace-gas retrievals including CO2. Here, we demonstrate that fluorescence information can be derived from space using potentially lower-cost hyperspectral instrumentation, i.e., more than an order of magnitude less spectral resolution than GOSAT, with a relatively simple algorithm. As a demonstration, we use the filling-in of one of the few wide and deep solar Fraunhofer lines in the red and far-red chlorophylla fluorescence bands, the calcium II line near 866 nm, to retrieve fluorescence with the SCanning Imaging Absorption spectroMeter for Atmospheric CHartographY (SCIAMACHY) satellite instrument. Although the signal from vegetation fluorescence is extremely weak at 866 nm, our results suggest that useful information may be obtained after adjustments are made to the observed spectra to correct for instrumental artifacts. We compare fluorescence from SCIAMACHY with that retrieved at 758 and 770 nm from similarly-corrected GOSAT data as well with the Enhanced Vegetation Index (EVI) from the MODerate-resolution Imaging Spectroradiometer (MODIS). We also show that filling-in occurs at 866 nm over barren areas, possibly originating from luminescent minerals in rock and soil.

Bi-fluorescence imaging for estimating accurately the nuclear condition of Rhizoctonia spp.

USDA-ARS?s Scientific Manuscript database

Aims: To simplify the determination of the nuclear condition of the pathogenic Rhizoctonia, which currently needs to be performed either using two fluorescent dyes, thus is more costly and time-consuming, or using only one fluorescent dye, and thus less accurate. Methods and Results: A red primary ...

Autofluorescence study and selected cyanidin quantification in the Jewel orchids Anoectochilus sp. and Ludisia discolor

PubMed Central

Poobathy, Ranjetta; Zakaria, Rahmad; Murugaiyah, Vikneswaran

2018-01-01

Anoectochilus sp. and Ludisia discolor are known as Jewel orchids. Both species are terrestrial wild orchids that grow in shaded areas of forests. The Jewel orchids are renowned for the beauty of their leaves, which are dark-green laced with silvery or golden veins. The orchids are used as a cure in various parts of Asia. Overharvesting and anthropogenic disturbances threaten the existence of the Jewel orchids in the wild, necessitating human intervention in their survival. An understanding of the structure and adaptations of a plant may assist in its survival when propagated outside of its habitat. In this study, ex vitro leaves of Anoectochilus sp. and L. discolor were subjected to freehand sectioning, and then inspected through brightfield and fluorescence microscopy. The study indicated that all parts of both plants presented typical monocotyledonous characteristics except the leaves. The leaves displayed dorsiventrality with distinct palisade and spongy mesophyll layers. The spongy mesophyll layer contained cells which fluoresced a bright red when exposed to ultraviolet, blue, and green light wavelengths, hinting at the presence of anthocyanins for photoprotection. Cyanidin was detected in the leaves of L. discolor, as enumerated through high performance liquid chromatography (HPLC). The observations indicated that Anoectochilus sp. and L. discolor are well-adapted to live under shaded conditions with minimal exposure to light. PMID:29649288

DOE Office of Scientific and Technical Information (OSTI.GOV)

Pletnev, Sergei; Shcherbo, Dmitry; Chudakov, Dmitry M.

The far-red fluorescent protein mKate {lambda}{sup ex}, 588 nm; {lambda}{sub em}, 635 nm; chromophore-forming triad Met{sup 63}-Tyr{sup 64}-Gly{sup 65}, originating from wild-type red fluorescent progenitor eqFP578 (sea anemone Entacmaea quadricolor), is monomeric and characterized by the pronounced pH dependence of fluorescence, relatively high brightness, and high photostability. The protein has been crystallized at a pH ranging from 2 to 9 in three space groups, and four structures have been determined by x-ray crystallography at the resolution of 1.75--2.6 {angstrom}. The pH-dependent fluorescence of mKate has been shown to be due to reversible cis-trans isomerization of the chromophore phenolic ring. Inmore » the non-fluorescent state at pH 2.0, the chromophore of mKate is in the trans-isomeric form. The weakly fluorescent state of the protein at pH 4.2 is characterized by a mixture of trans and cis isomers. The chromophore in a highly fluorescent state at pH 7.0/9.0 adopts the cis form. Three key residues, Ser{sup 143}, Leu{sup 174}, and Arg{sup 197} residing in the vicinity of the chromophore, have been identified as being primarily responsible for the far-red shift in the spectra. A group of residues consisting of Val{sup 93}, Arg{sup 122}, Glu{sup 155}, Arg{sup 157}, Asp{sup 159}, His{sup 169}, Ile{sup 171}, Asn{sup 173}, Val{sup 192}, Tyr{sup 194}, and Val{sup 216}, are most likely responsible for the observed monomeric state of the protein in solution.« less

Photo-convertible fluorescent proteins as tools for fresh insights on subcellular interactions in plants.

PubMed

Griffiths, N; Jaipargas, E-A; Wozny, M R; Barton, K A; Mathur, N; Delfosse, K; Mathur, J

2016-08-01

Optical highlighters comprise photo-activatable, photo-switchable and photo-convertible fluorescent proteins and are relatively recent additions to the toolbox utilized for live cell imaging research. Here, we provide an overview of four photo-convertible fluorescent proteins (pcFP) that are being used in plant cell research: Eos, Kaede, Maple and Dendra2. Each of these proteins has a significant advantage over other optical highlighters since their green fluorescent nonconverted forms and red fluorescent converted forms are generally clearly visible at expression levels that do not appear to interfere with subcellular dynamics and plant development. These proteins have become increasingly useful for understanding the role of transient and sustained interactions between similar organelles. Tracking of single organelles after green-to-red conversion has provided novel insights on plastids and their stroma-filled extensions and on the formation of mega-mitochondria. Similarly colour recovery after photo-conversion has permitted the estimation of nuclear endo-reduplication events and is being developed further to image protein trafficking within the lumen of the endoplasmic reticulum. We have also applied photo-convertible proteins to create colour-differentiation between similar cell types to follow their development. Both the green and red fluorescent forms of these proteins are compatible with other commonly used single coloured FPs. This has allowed us to develop simultaneous visualization schemes for up to five types of organelles and investigate organelle interactivity. The advantages and caveats associated with the use of photo-convertible fluorescent proteins are discussed. © 2016 The Authors Journal of Microscopy © 2016 Royal Microscopical Society.

Dual-Color Fluorescence Imaging to Monitor CYP3A4 and CYP3A7 Expression in Human Hepatic Carcinoma HepG2 and HepaRG Cells

PubMed Central

Kubiura, Musashi; Hayashi, Ayaka; Ohbayashi, Tetsuya; Kazuki, Yasuhiro; Chesné, Christophe; Oshimura, Mitsuo; Tada, Masako

2014-01-01

Human adult hepatocytes expressing CYP3A4, a major cytochrome P450 enzyme, are required for cell-based assays to evaluate the potential risk of drug-drug interactions caused by transcriptional induction of P450 enzymes in early-phase drug discovery and development. However, CYP3A7 is preferentially expressed in premature hepatoblasts and major hepatic carcinoma cell lines. The human hepatocellular carcinoma cell line HepaRG possesses a high self-renewal capacity and can differentiate into hepatic cells similar to human adult hepatocytes in vitro. Transgenic HepaRG cells, in which the expression of fluorescent reporters is regulated by 35 kb regulatory elements of CYP3A4, have a distinct advantage over human hepatocytes isolated by collagenase perfusion, which are unstable in culture. Thus, we created transgenic HepaRG and HepG2 cells by replacing the protein-coding regions of human CYP3A4 and CYP3A7 with enhanced green fluorescent protein (EGFP) and DsRed reporters, respectively, in a bacterial artificial chromosome vector that included whole regulatory elements. The intensity of DsRed fluorescence was initially high during the proliferation of transgenic HepaRG cells. However, most EGFP-positive cells were derived from those in which DsRed fluorescence was extinguished. Comparative analyses in these transgenic clones showed that changes in the total fluorescence intensity of EGFP reflected fold changes in the mRNA level of endogenous CYP3A4. Moreover, CYP3A4 induction was monitored by the increase in EGFP fluorescence. Thus, this assay provides a real-time evaluation system for quality assurance of hepatic differentiation into CYP3A4-expressing cells, unfavourable CYP3A4 induction, and fluorescence-activated cell sorting-mediated enrichment of CYP3A4-expressing hepatocytes based on the total fluorescence intensities of fluorescent reporters, without the need for many time-consuming steps. PMID:25101946

An epifluorescent attachment improves whole-plant digital photography of Arabidopsis thaliana expressing red-shifted green fluorescent protein

PubMed Central

Baker, Stokes S.; Vidican, Cleo B.; Cameron, David S.; Greib, Haittam G.; Jarocki, Christine C.; Setaputri, Andres W.; Spicuzza, Christopher H.; Burr, Aaron A.; Waqas, Meriam A.; Tolbert, Danzell A.

2012-01-01

Background and aims Studies have shown that levels of green fluorescent protein (GFP) leaf surface fluorescence are directly proportional to GFP soluble protein concentration in transgenic plants. However, instruments that measure GFP surface fluorescence are expensive. The goal of this investigation was to develop techniques with consumer digital cameras to analyse GFP surface fluorescence in transgenic plants. Methodology Inexpensive filter cubes containing machine vision dichroic filters and illuminated with blue light-emitting diodes (LED) were designed to attach to digital single-lens reflex (SLR) camera macro lenses. The apparatus was tested on purified enhanced GFP, and on wild-type and GFP-expressing arabidopsis grown autotrophically and heterotrophically. Principal findings Spectrum analysis showed that the apparatus illuminates specimens with wavelengths between ∼450 and ∼500 nm, and detects fluorescence between ∼510 and ∼595 nm. Epifluorescent photographs taken with SLR digital cameras were able to detect red-shifted GFP fluorescence in Arabidopsis thaliana leaves and cotyledons of pot-grown plants, as well as roots, hypocotyls and cotyledons of etiolated and light-grown plants grown heterotrophically. Green fluorescent protein fluorescence was detected primarily in the green channel of the raw image files. Studies with purified GFP produced linear responses to both protein surface density and exposure time (H0: β (slope) = 0 mean counts per pixel (ng s mm−2)−1, r2 > 0.994, n = 31, P < 1.75 × 10−29). Conclusions Epifluorescent digital photographs taken with complementary metal-oxide-semiconductor and charge-coupled device SLR cameras can be used to analyse red-shifted GFP surface fluorescence using visible blue light. This detection device can be constructed with inexpensive commercially available materials, thus increasing the accessibility of whole-organism GFP expression analysis to research laboratories and teaching institutions with small budgets. PMID:22479674

Global Monitoring of Terrestrial Chlorophyll Fluorescence from Moderate-spectral-resolution Near-infrared Satellite Measurements: Methodology, Simulations, and Application to GOME-2

NASA Technical Reports Server (NTRS)

Joiner, J.; Gaunter, L.; Lindstrot, R.; Voigt, M.; Vasilkov, A. P.; Middleton, E. M.; Huemmrich, K. F.; Yoshida, Y.; Frankenberg, C.

2013-01-01

Globally mapped terrestrial chlorophyll fluorescence retrievals are of high interest because they can provide information on the functional status of vegetation including light-use efficiency and global primary productivity that can be used for global carbon cycle modeling and agricultural applications. Previous satellite retrievals of fluorescence have relied solely upon the filling-in of solar Fraunhofer lines that are not significantly affected by atmospheric absorption. Although these measurements provide near-global coverage on a monthly basis, they suffer from relatively low precision and sparse spatial sampling. Here, we describe a new methodology to retrieve global far-red fluorescence information; we use hyperspectral data with a simplified radiative transfer model to disentangle the spectral signatures of three basic components: atmospheric absorption, surface reflectance, and fluorescence radiance. An empirically based principal component analysis approach is employed, primarily using cloudy data over ocean, to model and solve for the atmospheric absorption. Through detailed simulations, we demonstrate the feasibility of the approach and show that moderate-spectral-resolution measurements with a relatively high signal-to-noise ratio can be used to retrieve far-red fluorescence information with good precision and accuracy. The method is then applied to data from the Global Ozone Monitoring Instrument 2 (GOME-2). The GOME-2 fluorescence retrievals display similar spatial structure as compared with those from a simpler technique applied to the Greenhouse gases Observing SATellite (GOSAT). GOME-2 enables global mapping of far-red fluorescence with higher precision over smaller spatial and temporal scales than is possible with GOSAT. Near-global coverage is provided within a few days. We are able to show clearly for the first time physically plausible variations in fluorescence over the course of a single month at a spatial resolution of 0.5 deg × 0.5 deg. We also show some significant differences between fluorescence and coincident normalized difference vegetation indices (NDVI) retrievals.

Global Monitoring of Terrestrial Chlorophyll Fluorescence from Moderate-Spectral-Resolution Near-Infrared Satellite Measurements: Methodology, Simulations, and Application to GOME-2

NASA Technical Reports Server (NTRS)

Joiner, J.; Guanter, L.; Lindstrot, R.; Voigt, M.; Vasilkov, A. P.; Middleton, E. M.; Huemmrich, K. F.; Yoshida, Y.; Frankenberg, C.

2013-01-01

Globally mapped terrestrial chlorophyll fluorescence retrievals are of high interest because they can provide information on the functional status of vegetation including light-use efficiency and global primary productivity that can be used for global carbon cycle modeling and agricultural applications. Previous satellite retrievals of fluorescence have relied solely upon the filling-in of solar Fraunhofer lines that are not significantly affected by atmospheric absorption. Although these measurements provide near-global coverage on a monthly basis, they suffer from relatively low precision and sparse spatial sampling. Here, we describe a new methodology to retrieve global far-red fluorescence information; we use hyperspectral data with a simplified radiative transfer model to disentangle the spectral signatures of three basic components: atmospheric absorption, surface reflectance, and fluorescence radiance. An empirically based principal component analysis approach is employed, primarily using cloudy data over ocean, to model and solve for the atmospheric absorption. Through detailed simulations, we demonstrate the feasibility of the approach and show that moderate-spectral-resolution measurements with a relatively high signal-to-noise ratio can be used to retrieve far-red fluorescence information with good precision and accuracy. The method is then applied to data from the Global Ozone Monitoring Instrument 2 (GOME-2). The GOME-2 fluorescence retrievals display similar spatial structure as compared with those from a simpler technique applied to the Greenhouse gases Observing SATellite (GOSAT). GOME-2 enables global mapping of far-red fluorescence with higher precision over smaller spatial and temporal scales than is possible with GOSAT. Near-global coverage is provided within a few days. We are able to show clearly for the first time physically plausible variations in fluorescence over the course of a single month at a spatial resolution of 0.5 0.5. We also show some significant differences between fluorescence and coincident normalized difference vegetation indices (NDVI) retrievals.

Dual-color fluorescence imaging to monitor CYP3A4 and CYP3A7 expression in human hepatic carcinoma HepG2 and HepaRG cells.

PubMed

Tsuji, Saori; Kawamura, Fumihiko; Kubiura, Musashi; Hayashi, Ayaka; Ohbayashi, Tetsuya; Kazuki, Yasuhiro; Chesné, Christophe; Oshimura, Mitsuo; Tada, Masako

2014-01-01

Human adult hepatocytes expressing CYP3A4, a major cytochrome P450 enzyme, are required for cell-based assays to evaluate the potential risk of drug-drug interactions caused by transcriptional induction of P450 enzymes in early-phase drug discovery and development. However, CYP3A7 is preferentially expressed in premature hepatoblasts and major hepatic carcinoma cell lines. The human hepatocellular carcinoma cell line HepaRG possesses a high self-renewal capacity and can differentiate into hepatic cells similar to human adult hepatocytes in vitro. Transgenic HepaRG cells, in which the expression of fluorescent reporters is regulated by 35 kb regulatory elements of CYP3A4, have a distinct advantage over human hepatocytes isolated by collagenase perfusion, which are unstable in culture. Thus, we created transgenic HepaRG and HepG2 cells by replacing the protein-coding regions of human CYP3A4 and CYP3A7 with enhanced green fluorescent protein (EGFP) and DsRed reporters, respectively, in a bacterial artificial chromosome vector that included whole regulatory elements. The intensity of DsRed fluorescence was initially high during the proliferation of transgenic HepaRG cells. However, most EGFP-positive cells were derived from those in which DsRed fluorescence was extinguished. Comparative analyses in these transgenic clones showed that changes in the total fluorescence intensity of EGFP reflected fold changes in the mRNA level of endogenous CYP3A4. Moreover, CYP3A4 induction was monitored by the increase in EGFP fluorescence. Thus, this assay provides a real-time evaluation system for quality assurance of hepatic differentiation into CYP3A4-expressing cells, unfavourable CYP3A4 induction, and fluorescence-activated cell sorting-mediated enrichment of CYP3A4-expressing hepatocytes based on the total fluorescence intensities of fluorescent reporters, without the need for many time-consuming steps.

Intravital imaging of a pulmonary endothelial surface layer in a murine sepsis model.

PubMed

Park, Inwon; Choe, Kibaek; Seo, Howon; Hwang, Yoonha; Song, Eunjoo; Ahn, Jinhyo; Hwan Jo, You; Kim, Pilhan

2018-05-01

Direct intravital imaging of an endothelial surface layer (ESL) in pulmonary microcirculation could be a valuable approach to investigate the role of a vascular endothelial barrier in various pathological conditions. Despite its importance as a marker of endothelial cell damage and impairment of the vascular system, in vivo visualization of ESL has remained a challenging technical issue. In this work, we implemented a pulmonary microcirculation imaging system integrated to a custom-design video-rate laser scanning confocal microscopy platform. Using the system, a real-time cellular-level microscopic imaging of the lung was successfully performed, which facilitated a clear identification of individual flowing erythrocytes in pulmonary capillaries. Subcellular level pulmonary ESL was identified in vivo by fluorescence angiography using a dextran conjugated fluorophore to label blood plasma and the red blood cell (RBC) exclusion imaging analysis. Degradation of ESL width was directly evaluated in a murine sepsis model in vivo , suggesting an impairment of pulmonary vascular endothelium and endothelial barrier dysfunction.

Intravital imaging of a pulmonary endothelial surface layer in a murine sepsis model

PubMed Central

Park, Inwon; Choe, Kibaek; Seo, Howon; Hwang, Yoonha; Song, Eunjoo; Ahn, Jinhyo; Hwan Jo, You; Kim, Pilhan

2018-01-01

Direct intravital imaging of an endothelial surface layer (ESL) in pulmonary microcirculation could be a valuable approach to investigate the role of a vascular endothelial barrier in various pathological conditions. Despite its importance as a marker of endothelial cell damage and impairment of the vascular system, in vivo visualization of ESL has remained a challenging technical issue. In this work, we implemented a pulmonary microcirculation imaging system integrated to a custom-design video-rate laser scanning confocal microscopy platform. Using the system, a real-time cellular-level microscopic imaging of the lung was successfully performed, which facilitated a clear identification of individual flowing erythrocytes in pulmonary capillaries. Subcellular level pulmonary ESL was identified in vivo by fluorescence angiography using a dextran conjugated fluorophore to label blood plasma and the red blood cell (RBC) exclusion imaging analysis. Degradation of ESL width was directly evaluated in a murine sepsis model in vivo, suggesting an impairment of pulmonary vascular endothelium and endothelial barrier dysfunction. PMID:29760995

Archive of bacterial community in anhydrite crystals from a deep-sea basin provides evidence of past oil-spilling in a benthic environment in the Red Sea

NASA Astrophysics Data System (ADS)

Wang, Yong; Li, Tie Gang; Wang, Meng Ying; Lai, Qi Liang; Li, Jiang Tao; Gao, Zhao Ming; Shao, Zong Ze; Qian, Pei-Yuan

2016-11-01

In deep-sea sediment, the microbes present in anhydrite crystals are potential markers of the past environment. In the Atlantis II Deep, anhydrite veins were produced by mild mixture of calcium-rich hydrothermal solutions and sulfate in the bottom water, which had probably preserved microbial inhabitants in the past seafloor of the Red Sea. In this study, this hypothesis was tested by analyzing the metagenome of an anhydrite crystal sample from the Atlantis II Deep. The estimated age of the anhydrite layer was between 750 and 770 years, which might span the event of hydrothermal eruption into the benthic floor. The 16S/18S rRNA genes in the metagenome were assigned to bacteria, archaea, fungi and even invertebrate species. The dominant species in the crystals was an oil-degrading Alcanivorax borkumensis bacterium, which was not detected in the adjacent sediment layer. Fluorescence microscopy using 16S rRNA and marker gene probes revealed intact cells of the Alcanivorax bacterium in the crystals. A draft genome of A. borkumensis was binned from the metagenome. It contained all functional genes for alkane utilization and the reduction of nitrogen oxides. Moreover, the metagenomes of the anhydrites and control sediment contained aromatic degradation pathways, which were mostly derived from Ochrobactrum sp. Altogether, these results indicate an oxic, oil-spilling benthic environment in the Atlantis II basin of the Red Sea in approximately the 14th century. The original microbial inhabitants probably underwent a dramatic selection process via drastic environmental changes following the formation of an overlying anoxic brine pool in the basin due to hydrothermal activities.

Facile and green synthesis of (La0.95Eu0.05)2O2S red phosphors with sulfate-ion pillared layered hydroxides as a new type of precursor: controlled hydrothermal processing, phase evolution and photoluminescence

PubMed Central

Wang, Xuejiao; Li, Ji-Guang; Zhu, Qi; Li, Xiaodong; Sun, Xudong; Sakka, Yoshio

2014-01-01

This study presents a facile and green route for the synthesis of (La0.95Eu0.05)2O2S red phosphors of controllable morphologies, with the sulfate-type layered hydroxides of Ln2(OH)4SO4·2H2O (Ln = La and Eu) as a new type of precursor. The technique takes advantage of the fact that the precursor has had the exact Ln:S molar ratio of the targeted phosphor, thus saving the hazardous sulfurization reagents indispensable to traditional synthesis. Controlled hydrothermal processing at 120 °C yielded phase-pure Ln2(OH)4SO4·2H2O crystallites in the form of either nanoplates or microprisms, which can both be converted into Ln2O2S phosphor via a Ln2O2SO4 intermediate upon annealing in flowing H2 at a minimum temperature of ∼ 700 °C. The nanoplates collapse into relatively rounded Ln2O2S particles while the microprisms retain well their initial morphologies at 1 200 °C, thus yielding two types of red phosphors. Photoluminescence excitation (PLE) studies found two distinct charge transfer (CT) excitation bands of O2− → Eu3+ at ∼ 270 nm and S2− → Eu3+ at ∼ 340 nm for the Ln2O2S phosphors, with the latter being stronger and both significantly stronger than the intrinsic intra-f transitions of Eu3+. The two types of phosphors share high similarities in the positions of PLE/PL (photoluminescence) bands and both show the strongest red emission at 627 nm (5D0 → 7F2 transition of Eu3+) under S2− → Eu3+ CT excitation at 340 nm. The PLE/PL intensities show clear dependence on particle morphology and calcination temperature, which were investigated in detail. Fluorescence decay analysis reveals that the 627 nm red emission has a lifetime of ∼ 0.5 ms for both types of the phosphors. PMID:27877631

Depth-resolved fluorescence of biological tissue

NASA Astrophysics Data System (ADS)

Wu, Yicong; Xi, Peng; Cheung, Tak-Hong; Yim, So Fan; Yu, Mei-Yung; Qu, Jianan Y.

2005-06-01

The depth-resolved autofluorescence ofrabbit oral tissue, normal and dysplastic human ectocervical tissue within l20μm depth were investigated utilizing a confocal fluorescence spectroscopy with the excitations at 355nm and 457nm. From the topmost keratinizing layer of oral and ectocervical tissue, strong keratin fluorescence with the spectral characteristics similar to collagen was observed. The fluorescence signal from epithelial tissue between the keratinizing layer and stroma can be well resolved. Furthermore, NADH and FADfluorescence measured from the underlying non-keratinizing epithelial layer were strongly correlated to the tissue pathology. This study demonstrates that the depth-resolved fluorescence spectroscopy can reveal fine structural information on epithelial tissue and potentially provide more accurate diagnostic information for determining tissue pathology.

Photoelectron Transfer at ZnTPyP Self-Assembly/TiO2 Interfaces for Enhanced Two-Photon Photodynamic Therapy.

PubMed

Liu, Yanyan; Meng, Xianfu; Wang, Han; Tang, Zhongmin; Zuo, Changjing; He, Mingyuan; Bu, Wenbo

2018-01-17

Two-photon (TP) absorption nanomaterials are highly desirable for deep-tissue clinical diagnostics and orthotopic disease treatment. Here, a well-designed core/shell nanostructure was successfully synthesized with a ZnTPyP self-assembly nanocrystal (ZSN) inner core coated by a homogeneous TiO 2 layer outside (ZSN-TO). The ZSN is a good photosemiconductor, showing both one-photon (OP) and TP absorption properties for red fluorescence emission and electron-hole pair generation; TiO 2 with good biocompatibility acts as the electron acceptor, which can transfer photoelectron from ZSN to TiO 2 for highly effective electron-hole separation, favoring the production of long-life superoxide anion (O 2 •- ) by electrons and oxygen and strong oxidizing hydroxyl radical (•OH) by holes and surrounding H 2 O. Once pretreated with ZSN-TO, the simultaneous OP-405 nm or TP-800 nm laser stimulation and fluorescent imaging of reactive oxygen species (ROS) showed dynamical and continuous generation of ROS in HeLa cells, with cytotoxicity significantly increasing via the type-1-like photodynamic therapy process. The results demonstrated that the combination of organic ZSN with inorganic TiO 2 has great applications as an excellent photosensitizer for deep-tissue fluorescent imaging and noninvasive disease treatment via TP photodynamic therapy.

A Red-Emitting, Multidimensional Sensor for the Simultaneous Cellular Imaging of Biothiols and Phosphate Ions †

PubMed Central

Herrero-Foncubierta, Pilar; Cuerva, Juan M.; Miguel, Delia

2018-01-01

The development of new fluorescent probes for cellular imaging is currently a very active field because of the large potential in understanding cell physiology, especially targeting anomalous behaviours due to disease. In particular, red-emitting dyes are keenly sought, as the light in this spectral region presents lower interferences and a deeper depth of penetration in tissues. In this work, we have synthesized a red-emitting, dual probe for the multiplexed intracellular detection of biothiols and phosphate ions. We have prepared a fluorogenic construct involving a silicon-substituted fluorescein for red emission. The fluorogenic reaction is selectively started by the presence of biothiols. In addition, the released fluorescent moiety undergoes an excited-state proton transfer reaction promoted by the presence of phosphate ions, which modulates its fluorescence lifetime, τ, with the total phosphate concentration. Therefore, in a multidimensional approach, the intracellular levels of biothiols and phosphate can be detected simultaneously using a single fluorophore and with spectral clearing of cell autofluorescence interferences. We have applied this concept to different cell lines, including photoreceptor cells, whose levels of biothiols are importantly altered by light irradiation and other oxidants. PMID:29315248

Ratiometric analysis of Acridine Orange staining in the study of acidic organelles and autophagy.

PubMed

Thomé, Marcos P; Filippi-Chiela, Eduardo C; Villodre, Emilly S; Migliavaca, Celina B; Onzi, Giovana R; Felipe, Karina B; Lenz, Guido

2016-12-15

Acridine Orange is a cell-permeable green fluorophore that can be protonated and trapped in acidic vesicular organelles (AVOs). Its metachromatic shift to red fluorescence is concentration-dependent and, therefore, Acridine Orange fluoresces red in AVOs, such as autolysosomes. This makes Acridine Orange staining a quick, accessible and reliable method to assess the volume of AVOs, which increases upon autophagy induction. Here, we describe a ratiometric analysis of autophagy using Acridine Orange, considering the red-to-green fluorescence intensity ratio (R/GFIR) to quantify flow cytometry and fluorescence microscopy data of Acridine-Orange-stained cells. This method measured with accuracy the increase in autophagy induced by starvation or rapamycin, and the reduction in autophagy produced by bafilomycin A1 or the knockdown of Beclin1 or ATG7. Results obtained with Acridine Orange, considering R/GFIR, correlated with the conversion of the unlipidated form of LC3 (LC3-I) into the lipidated form (LC3-II), SQSTM1 degradation and GFP-LC3 puncta formation, thus validating this assay to be used as an initial and quantitative method for evaluating the late step of autophagy in individual cells, complementing other methods. © 2016. Published by The Company of Biologists Ltd.

Synthesis and optical properties of water-soluble biperylene-based dendrimers.

PubMed

Shao, Pin; Jia, Ningyang; Zhang, Shaojuan; Bai, Mingfeng

2014-05-30

We report the synthesis and photophysical properties of three biperylene-based dendrimers, which show red fluorescence in water. A fluorescence microscopy study demonstrated uptake of biperylene-based dendrimers in living cells. Our results indicate that these biperylene-based dendrimers are promising candidates in fluorescence imaging applications with the potential as therapeutic carriers.

Deeper Insight into Fluorescence-Excitation of Molecules by Light

ERIC Educational Resources Information Center

Wahab, M. Farooq; Gore, Gordon R.

2013-01-01

In a recent issue of "TPT," Gordon Gore made interesting observations about the red or yellow fluorescence when laser beams are passed through olive oil. With the excellent visuals contained in that article, we present a pictorial explanation of the questions that were raised in Ref 1 ("Fun with Fluorescence in Olive Oil,"…

Mass spectrometric imaging of red fluorescent protein in breast tumor xenografts.

PubMed

Chughtai, Kamila; Jiang, Lu; Post, Harm; Winnard, Paul T; Greenwood, Tiffany R; Raman, Venu; Bhujwalla, Zaver M; Heeren, Ron M A; Glunde, Kristine

2013-05-01

Mass spectrometric imaging (MSI) in combination with electrospray mass spectrometry (ESI-MS) is a powerful technique for visualization and identification of a variety of different biomolecules directly from thin tissue sections. As commonly used tools for molecular reporting, fluorescent proteins are molecular reporter tools that have enabled the elucidation of a multitude of biological pathways and processes. To combine these two approaches, we have performed targeted MS analysis and MALDI-MSI visualization of a tandem dimer (td)Tomato red fluorescent protein, which was expressed exclusively in the hypoxic regions of a breast tumor xenograft model. For the first time, a fluorescent protein has been visualized by both optical microscopy and MALDI-MSI. Visualization of tdTomato by MALDI-MSI directly from breast tumor tissue sections will allow us to simultaneously detect and subsequently identify novel molecules present in hypoxic regions of the tumor. MS and MALDI-MSI of fluorescent proteins, as exemplified in our study, is useful for studies in which the advantages of MS and MSI will benefit from the combination with molecular approaches that use fluorescent proteins as reporters.

"siRNA traffic lights": arabino-configured 2'-anchors for fluorescent dyes are key for dual color readout in cell imaging.

PubMed

Steinmeyer, Jeannine; Walter, Heidi-Kristin; Bichelberger, Mathilde A; Schneider, Violetta; Kubař, Tomáš; Rönicke, Franziska; Olshausen, Bettina; Nienhaus, Karin; Nienhaus, Gerd Ulrich; Schepers, Ute; Elstner, Marcus; Wagenknecht, Hans-Achim

2018-05-23

Two fluorescent dyes covalently attached in diagonal interstrand orientation to siRNA undergo energy transfer and thereby enable a dual color fluorescence readout (red/green) for hybridization. Three different structural variations were carried out and compared by their optical properties, including (i) the base surrogate approach with an acyclic linker as a substitute of the 2-deoxyriboside between the phosphodiester bridges, (ii) the 2'-modification of conventional ribofuranosides and (iii) the arabino-configured 2'-modification. The double stranded siRNA with the latter type of modification delivered the best energy transfer efficiency, which was explained by molecular dynamics simulations that showed that the two dyes are more flexible at the arabino-configured sugars compared to the completely stacked situation at the ribo-configured ones. Single molecule fluorescence lifetime measurements indicate their application in fluorescence cell imaging, which reveals a red/green fluorescence contrast in particular for the arabino-configured 2'-modification by the two dyes, which is key for tracking of siRNA transport into HeLa cells.

Photodynamic effects on human and chicken erythrocytes

NASA Astrophysics Data System (ADS)

Kimel, Sol; Koenig, Karsten; Berns, Michael W.

1995-02-01

The intracellular accumulation of a variety of photosensitizers in human (non-nucleated) and chicken (nucleated) erythrocytes, as well as the photodynamically induced hemolysis were studied using 488 nm laser microirradiation (15 (mu) W, 100X) and confocal laser scanning fluorescence microscopy. Cells incubated with the negatively charged hydrophilic compounds TPPS4 and Pd-TPPS4 exhibited no significant fluorescence before irradiation, but developed strong fluorescence in the cellular and nuclear membranes following photoinduced membrane damage. In contrast, microirradiation of Photofrin-incubated erythrocytes showed instantaneous fluorescence which decreased due to photodegradation. For the cationic, hydrophilic dye Methylene Blue, significant fluorescence was detected in the nucleus only. Following ALA incubation, large intercellular differences were observed in fluorescence in the red spectral region. These differences are probably due to the differential ability of individual erythrocytes to biosynthesize protoporphyrin IX. Photofrin was the most efficient photosensitizer to induce hemolysis. Higher radiant exposures were required for lysis of nucleated than of human red blood cells, except in the case of Methylene Blue. Irradiation was more efficient for unwashed cell suspensions than for washed suspensions, indicating the non-negligible role of extracellular photosensitizing molecules.

Teaching laser-induced fluorescence of plant leaves

NASA Astrophysics Data System (ADS)

Lenk, Sándor; Gádoros, Patrik; Kocsányi, László; Barócsi, Attila

2016-11-01

Plants convert carbon dioxide into sugars using the energy of sunlight. Absorbed light unused for conversion is dissipated primarily as heat with a small fraction re-emitted as fluorescence at longer wavelengths. One can use the latter to estimate photosynthetic activity. The illumination of intact leaves with strong light after keeping them in dark for tens of minutes results in a rapid increase followed by a slow decay of fluorescence emission from the fluorophore chlorophyll-a, called the Kautsky effect. This paper describes a laboratory practice that introduces students of physics or engineering into this research field. It begins with the spectral measurement of the fluorescence emitted by a plant leaf upon UV excitation. Then it focuses on the red and far-red components of the fluorescence emission spectrum characteristic to the chlorophyll-a molecule and presents an inexpensive demonstration of the Kautsky effect. As researchers use more complex measurement techniques and tools, the practice ends up with the demonstration of an intelligent fluorosensor, a compact tool developed for plant physiological research and horticulture applications together with a brief interpretation of some important fluorescence parameters.

Toluidine blue-O is a Nissl bright-field counterstain for lipophilic fluorescent tracers Di-ASP, DiI and DiO.

PubMed

Chelvanayagam, D K; Beazley, L D

1997-03-01

The stain toluidine blue-O (tol blue), applied to sections of neural tissue, is shown to be compatible with the vivid fluorescent lipophilic neural tracers 4-(4-dihexadecylaminostyryl)-N-methylpyridinium iodide (Di-ASP), 3,3'-dioctadecyloxacarbocyanine perchlorate (DiO) and 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI). As with other Nissl stains, toluidine blue-O fluoresces in the red end of the spectrum but such fluorescence quenches upon binding with tissue. Moreover, progressive staining occurs at concentrations low enough to minimise any background fluorescence attributable to non-specific residence of the stain. The bright yellow Di-ASP and vivid green DiO signals are spectrally removed from the red fluorescence of toluidine blue-O. With toluidine blue-O counterstaining, Di-ASP generally offers contrast superior to that with DiI, however, the latter is improved by viewing in a polarised green bright field. Visible Di-ASP emission, although broad, peaks at a more film-sensitive region of the spectrum than that for DiI, thus reducing the photographic exposure required.

Instrumentation in Developing Chlorophyll Fluorescence Biosensing: A Review

PubMed Central

Fernandez-Jaramillo, Arturo A.; Duarte-Galvan, Carlos; Contreras-Medina, Luis M.; Torres-Pacheco, Irineo; de J. Romero-Troncoso, Rene; Guevara-Gonzalez, Ramon G.; Millan-Almaraz, Jesus R.

2012-01-01

Chlorophyll fluorescence can be defined as the red and far-red light emitted by photosynthetic tissue when it is excited by a light source. This is an important phenomenon which permits investigators to obtain important information about the state of health of a photosynthetic sample. This article reviews the current state of the art knowledge regarding the design of new chlorophyll fluorescence sensing systems, providing appropriate information about processes, instrumentation and electronic devices. These types of systems and applications can be created to determine both comfort conditions and current problems within a given subject. The procedure to measure chlorophyll fluorescence is commonly split into two main parts; the first involves chlorophyll excitation, for which there are passive or active methods. The second part of the procedure is to closely measure the chlorophyll fluorescence response with specialized instrumentation systems. Such systems utilize several methods, each with different characteristics regarding to cost, resolution, ease of processing or portability. These methods for the most part include cameras, photodiodes and satellite images. PMID:23112686

Large Fluorescence Response by Alcohol from a Bis(benzoxazole)-Zinc(II) Complex: The Role of Excited State Intramolecular Proton Transfer

PubMed Central

Wang, Junfeng; Chu, Qinghui; Liu, Xiumin; Wesdemiotis, Chrys

2013-01-01

The formation of a bis(HBO) anion is known to turn-on the fluorescence to give red emission, via controlling the excited-state intramolecular proton transfer (ESIPT). The poor stability of the formed anion, however, hampered its application. The anion stability is found to be greatly improved by attaching the anion to Zn2+ cation (i.e. forming zinc complex), whose emission is at λem ≈ 550 and 760 nm. Interestingly, addition of methanol to the zinc complex induces a remarkable red fluorescence (λem ≈ 630 nm, ϕfl ≈ 0.8). With the aid of spectroscopic studies (1H NMR, UV-vis, fluorescence, and mass spectra), the structures of the zinc complexes are characterized. The emission species is identified as a dimer-like structure. The study thus reveals an effective fluorescence switching mechanism that could further advance the application of ESIPT-based sensors. PMID:23514312

A coumarin-derived Cu2 +-fluorescent chemosensor and its direct application in aqueous media

NASA Astrophysics Data System (ADS)

Mergu, Naveen; Kim, Myeongjin; Son, Young-A.

2018-01-01

A novel coumarin-based receptor bearing a benzohydrazide (FCBH) was developed as a fluorescent chemosensor with high selectivity toward Cu2 +. The sensor was successfully applied to the monitoring of Cu2 + in aqueous solution. After the addition of Cu2 + to FCBH, the color of the solution changed from greenish-yellow to red, and the absorption band at 457 nm red-shifted to 517 nm. The fluorescent green color of FCBH disappeared and the fluorescence emission was completely quenched in the presence of Cu2 +. Upon the addition of Cu2 +, deprotonation of FCBH occurred, and a 1:1 metal-ligand complex formed. DFT theoretical investigation was carried out to understand the behavior of the sensing probe toward Cu2 +. Additionally, the quenched fluorescence of the FCBH-Cu2 + complex was restored upon the addition of CN- ions. The possible sensing mechanism of FCBH toward Cu2 + was derived from experimental and theoretical examinations.

Highly Stable and Red-Emitting Nanovesicles Incorporating Lipophilic Diketopyrrolopyrroles for Cell Imaging.

PubMed

Veciana, Jaume; Ardizzone, Antonio; Blasi, Davide; Grimaldi, Natascia; Sala, Santi; Ratera, Imma; Vona, Danilo; Rosspeintner, Arnulf; Punzi, Angela; Altamura, Emiliano; Vauthey, Eric; Farinola, Gianluca M; Ventosa, Nora

2018-06-05

Diketopyrrolopyrroles (DPPs) have recently attracted large interest as highly bright and photostable red-emitting molecules. However, their tendency to form non-fluorescent aggregates in water via the so-called Aggregation Caused Quenching (ACQ) effect is a major issue that limits their application under the microscope. In this work, two DPP molecules have been incorporated in the membrane of highly stable and water-soluble Quatsomes (QS, nanovesicles made by surfactants and sterols), allowing their nanostructuration in water limiting at the same time the ACQ effect. The obtained fluorescent organic nanoparticles (FONs) showed superior structural homogeneity along with long-time colloidal and optical stability. A thorough one- (1P) and two-photon (2P) fluorescence characterization revealed the promising photophysical features of these fluorescent nanovesicles, which showed a high 1P and 2P brightness. Finally, the fluorescent QSs were used for the in vitro bioimaging of Saos-2 osteosarcoma cell lines, demonstrating their potential as nanomaterials for bioimaging applications. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Deriving chlorophyll fluorescence emissions of vegetation canopies from high resolution field reflectance spectra

NASA Astrophysics Data System (ADS)

Middleton, Elizabeth M.; Corp, Lawrence A.; Daughtry, Craig S.; Entcheva Campbell, Petya K.; Butcher, L. Maryn

2005-11-01

Fluorescence of foliage in the laboratory has proven more rigorous than reflectance for correlation to plant physiology. Especially useful are emissions produced from two stable red and far-red chlorophyll fluorescence (ChlF) peaks centered at 685 nm and 735 nm. Methods have been developed elsewhere to extract steady state solar induced fluorescence (SIF) from apparent reflectance of vegetation canopies/landscapes using the Fraunhofer Line Depth (FLD) principal. Our study utilized these methods in conjunction with field-acquired high spectral resolution canopy reflectance spectra obtained in 2004 and 2005 over corn crops and small tree plots of three deciduous species (red maple, tulip poplar, sweet gum). Leaf level measurements were also made of foliage which included ChlF, photosynthesis, and leaf constituents (photosynthetic pigment, carbon (C), and nitrogen (N) contents). As part of ongoing experiments, measurements were made on N application plots within corn (280, 140, 70, and 0 kg N/ha) and tree (0, 37.5, 75, 112.5, 150 kg N /ha) sites at the USDA/Agriculture Research Service in Beltsville, MD. SIF intensities for ChlF were derived directly from canopy reflectance spectra in specific narrow- band regions associated with atmospheric oxygen absorption features centered at 688 and 760 nm. The red/far-red SIF ratio (SIFratio) derived from these field reflectance spectra successfully discriminated foliar pigment ratios altered by N application rates in both corn crops. This ratio was also positively correlated to the C/N ratio at leaf and canopy levels, for the available corn data (e.g., 2004). No consistent N treatment or species differences in SIF were detected in the tree foliage, but additional 2005 data are forthcoming. This study has relevance to future passive satellite remote sensing approaches to monitoring C dynamics from space.

Refined crystal structure of DsRed, a red fluorescent protein from coral, at 2.0-A resolution.

PubMed

Yarbrough, D; Wachter, R M; Kallio, K; Matz, M V; Remington, S J

2001-01-16

The crystal structure of DsRed, a red fluorescent protein from a corallimorpharian, has been determined at 2.0-A resolution by multiple-wavelength anomalous dispersion and crystallographic refinement. Crystals of the selenomethionine-substituted protein have space group P2(1) and contain a tetramer with 222 noncrystallographic symmetry in the asymmetric unit. The refined model has satisfactory stereochemistry and a final crystallographic R factor of 0.162. The protein, which forms an obligatory tetramer in solution and in the crystal, is a squat rectangular prism comprising four protomers whose fold is extremely similar to that of the Aequorea victoria green fluorescent protein despite low ( approximately 23%) amino acid sequence homology. The monomer consists of an 11-stranded beta barrel with a coaxial helix. The chromophores, formed from the primary sequence -Gln-Tyr-Gly- (residues 66-68), are arranged in a approximately 27 x 34-A rectangular array in two approximately antiparallel pairs. The geometry at the alpha carbon of Gln-66 (refined without stereochemical restraints) is consistent with an sp(2) hybridized center, in accord with the proposal that red fluorescence is because of an additional oxidation step that forms an acylimine extension to the chromophore [Gross, L. A., Baird, G. S., Hoffman, R. C., Baldridge, K. K. & Tsien, R. Y. (2000) Proc. Natl. Acad. Sci. USA 87, 11990-11995]. The carbonyl oxygen of Phe-65 is almost 90 degrees out of the plane of the chromophore, consistent with theoretical calculations suggesting that this is the minimum energy conformation of this moiety despite the conjugation of this group with the rest of the chromophore.

Engineering ESPT pathways based on structural analysis of LSSmKate red fluorescent proteins with large Stokes shift.

PubMed

Piatkevich, Kiryl D; Malashkevich, Vladimir N; Almo, Steven C; Verkhusha, Vladislav V

2010-08-11

LSSmKate1 and LSSmKate2 are monomeric red fluorescent proteins (RFPs) with large Stokes shifts (LSSs), which allows for efficient separation of absorbance and emission maxima, as well as for excitation with conventional two-photon laser sources. These LSSmKates differ by a single amino acid substitution at position 160 and exhibit absorbance maxima around 460 nm, corresponding to a neutral DsRed-like chromophore. However, excitation at 460 nm leads to fluorescence emission above 600 nm. Structures of LSSmKate1 and LSSmKate2, determined at resolutions of 2.0 and 1.5 A, respectively, revealed that the predominant DsRed-chromophore configurations are cis for LSSmKate1 but trans for LSSmKate2. Crystallographic and mutagenesis analyses, as well as isotope and temperature dependences, suggest that an excited-state proton transfer (ESPT) is responsible for the LSSs observed in LSSmKates. Hydrogen bonding between the chromophore hydroxyl and Glu160 in LSSmKate1 and a proton relay involving the chromophore tyrosine hydroxyl, Ser158, and the Asp160 carboxylate in LSSmKate2 represent the putative ESPT pathways. Comparisons with mKeima LSS RFP suggest that similar proton relays could be engineered in other FPs. Accordingly, we mutated positions 158 and 160 in several conventional red-shifted FPs, including mNeptune, mCherry, mStrawberry, mOrange, and mKO, and the resulting FP variants exhibited LSS fluorescence emission in a wide range of wavelengths from 560 to 640 nm. These data suggest that different chromophores formed by distinct tripeptides in different environments can be rationally modified to yield RFPs with novel photochemical properties.

Photoabsorption of green and red fluorescent protein chromophore anions in vacuo.

PubMed

Wan, Songbo; Liu, Shasha; Zhao, Guangjiu; Chen, Maodu; Han, Keli; Sun, Mengtao

2007-09-01

Photoabsorption properties of green and red fluorescent protein chromophore anions in vacuo were investigated theoretically, based on the experimental results in gas phase [Phys. Rev. Lett. 2001, 87, 228102; Phys. Rev. Lett. 2003, 90, 118103]. Their calculated transition energies in absorption with TD-DFT and ZINDO methods are directly compared to the experimental reports in gas phase, and the calculations with ZINDO method can correctly reproduce the absorption spectra. The orientation and strength of their transition dipole moments were revealed with transition density. We also showed the orientation and result of their intramolecular charge transfer with transition difference density. The calculated results show that with the increase of the extended conjugated system, the orientation of transition dipole moments and the orientation of charge transfer can be reversed. They are the linear responds with the external electric fields. These theoretical results reveal the insight understanding of the photoinduced dynamics of green and red fluorescent protein chromophore anions and cations in vacuo.

An improved high-throughput Nile red fluorescence assay for estimating intracellular lipids in a variety of yeast species

PubMed Central

Sitepu, I.R.; Ignatia, L.; Franz, A. K.; Wong, D. M.; Faulina, S.A.; Tsui, M.; Kanti, A.; Boundy-Mills, K.

2012-01-01

A rapid and inexpensive method for estimating lipid content of yeasts is needed for screening large numbers of yeasts samples. Nile red is a fluorescent lipophilic dye used for detection and quantification of intracellular lipid droplets in various biological system including algae, yeasts and filamentous fungi. However, a published assay for yeast is affected by variable diffusion across the cell membrane, and variation in the time required to reach maximal fluorescence emission. In this study, parameters that may influence the emission were varied to determine optimal assay conditions. An improved assay with a high-throughput capability was developed that includes the addition of dimethyl sulfoxide (DMSO) solvent to improve cell permeability, elimination of the washing step, the reduction of Nile red concentration, kinetic readings rather than single time-point reading, and utilization of a black 96-well microplate. The improved method was validated by comparison to gravimetric determination of lipid content of a broad variety of ascomycete and basidiomycete yeast species. PMID:22985718

A Unique Collection of Palaeolithic Painted Portable Art: Characterization of Red and Yellow Pigments from the Parpalló Cave (Spain)

PubMed Central

Villaverde Bonilla, Valentín; Ródenas Marín, Isabel; Murcia Mascarós, Sonia

2016-01-01

In this work we analyze the pigments used in the decoration of red and yellow motifs present in the portable art of the Parpalló Cave (Gandía, Spain), one of the most important Palaeolithic sites in the Spanish Mediterranean region. Energy dispersive X-ray fluorescence spectrometry (EDXRF) and spectrophotometry in the visible region (CIEL*a*b*color coordinates and spectral reflectance curves) were used to perform in situ fast analyses of the red and yellow motifs with portable equipment and to characterize their elemental composition and their colorimetric perception, respectively. According to the elemental composition, the intensity of the fluorescence iron signals in red and yellow motifs are higher than average values in the rock substrates. As expected, red motifs possess high values of the chromatic coordinate a* and yellow motifs possess high values of b*. This characterization was complemented with FT-IR analyses of microsamples detached from the red and yellow colored zones of a small set of plaquettes. Our results show that the artists used red and yellow pigments in the decoration likely derived from natural iron oxides as hematite and goethite. PMID:27732605

A Unique Collection of Palaeolithic Painted Portable Art: Characterization of Red and Yellow Pigments from the Parpalló Cave (Spain).

PubMed

Roldán García, Clodoaldo; Villaverde Bonilla, Valentín; Ródenas Marín, Isabel; Murcia Mascarós, Sonia

2016-01-01

In this work we analyze the pigments used in the decoration of red and yellow motifs present in the portable art of the Parpalló Cave (Gandía, Spain), one of the most important Palaeolithic sites in the Spanish Mediterranean region. Energy dispersive X-ray fluorescence spectrometry (EDXRF) and spectrophotometry in the visible region (CIEL*a*b*color coordinates and spectral reflectance curves) were used to perform in situ fast analyses of the red and yellow motifs with portable equipment and to characterize their elemental composition and their colorimetric perception, respectively. According to the elemental composition, the intensity of the fluorescence iron signals in red and yellow motifs are higher than average values in the rock substrates. As expected, red motifs possess high values of the chromatic coordinate a* and yellow motifs possess high values of b*. This characterization was complemented with FT-IR analyses of microsamples detached from the red and yellow colored zones of a small set of plaquettes. Our results show that the artists used red and yellow pigments in the decoration likely derived from natural iron oxides as hematite and goethite.

Lettuce flavonoids screening and phenotyping by chlorophyll fluorescence excitation ratio.

PubMed

Zivcak, Marek; Brückova, Klaudia; Sytar, Oksana; Brestic, Marian; Olsovska, Katarina; Allakhverdiev, Suleyman I

2017-06-01

Environmentally induced variation and the genotypic differences in flavonoid and phenolic content in lettuce can be reliably detected using the appropriate parameters derived from the records of rapid non-invasive fluorescence technique. The chlorophyll fluorescence excitation ratio method was designed as a rapid and non-invasive tool to estimate the content of UV-absorbing phenolic compounds in plants. Using this technique, we have assessed the dynamics of accumulation of flavonoids related to developmental changes and environmental effects. Moreover, we have tested appropriateness of the method to identify the genotypic differences and fluctuations in total phenolics and flavonoid content in lettuce. Six green and two red genotypes of lettuce (Lactuca sativa L.) grown in pots were exposed to two different environments for 50 days: direct sunlight (UV-exposed) and greenhouse conditions (low UV). The indices based on the measurements of chlorophyll fluorescence after red, green and UV excitation indicated increase of the content of UV-absorbing compounds and anthocyanins in the epidermis of lettuce leaves. In similar, the biochemical analyses performed at the end of the experiment confirmed significantly higher total phenolic and flavonoid content in lettuce plants exposed to direct sun compared to greenhouse conditions and in red compared to green genotypes. As the correlation between the standard fluorescence indices and the biochemical records was negatively influenced by the presence of red genotypes, we proposed the use of a new parameter named Modified Flavonoid Index (MFI) taking into an account both absorbance changes due to flavonol and anthocyanin content, for which the correlation with flavonoid and phenolic content was relatively good. Thus, our results confirmed that the fluorescence excitation ratio method is useful for identifying the major differences in phenolic and flavonoid content in lettuce plants and it can be used for high-throughput pre-screening and phenotyping of leafy vegetables in research and breeding applications towards improvement of vegetable health effects.

Lipid chain saturation and the cholesterol in the phospholipid membrane affect the spectroscopic properties of lipophilic dye nile red

NASA Astrophysics Data System (ADS)

Halder, Animesh; Saha, Baishakhi; Maity, Pabitra; Kumar, Gopinatha Suresh; Sinha, Deepak Kumar; Karmakar, Sanat

2018-02-01

We have studied the effect of composition and the phase state of phospholipid membranes on the emission spectrum, anisotropy and lifetime of a lipophilic fluorescence probe nile red. Fluorescence spectrum of nile red in membranes containing cholesterol has also been investigated in order to get insights into the influence of cholesterol on the phospholipid membranes. Maximum emission wavelength (λem) of nile red in the fluid phase of saturated and unsaturated phospholipids was found to differ by 10 nm. The λem was also found to be independent of chain length and charge of the membrane. However, the λem is strongly dependent on the temperature in the gel phase. The λem and rotational diffusion rate decrease, whereas the anisotropy and lifetime increase markedly with increasing cholesterol concentration for saturated phosoholipids, such as, dimyristoyl phosphatidylcholine (DMPC) in the liquid ordered phase. However, these spectroscopic properties do not alter significantly in case of unsaturated phospholipids, such as, dioleoyl phosphatidylcholine (DOPC) in liquid disordered phase. Interestingly, red edge excitation shift (REES) in the presence of lipid-cholesterol membranes is the direct consequences of change in rotational diffusion due to motional restriction of lipids in the presence of cholesterol. This study provides correlations between the membrane compositions and fluorescence spectral features which can be utilized in a wide range of biophysical fields as well the cell biology.

Nitric oxide measurements in hTERT-RPE cells and subcellular fractions exposed to low levels of red light

NASA Astrophysics Data System (ADS)

Wigle, Jeffrey C.; Castellanos, Cherry C.; Denton, Michael L.; Holwitt, Eric A.

2014-02-01

Cells in a tissue culture model for laser eye injury exhibit increased resistance to a lethal pulse of 2.0-μm laser radiation if the cells are first exposed to 2.88 J/cm2 of red light 24 hr prior to the lethal laser exposure. Changes in expression of various genes associated with apoptosis have been observed, but the biochemical link between light absorption and gene expression remains unknown. Cytochome c oxidase (CCOX), in the electron transport chain, is the currentlyhypothesized absorber. Absorption of the red light by CCOX is thought to facilitate displacement of nitric oxide (NO) by O2 in the active site, increasing cellular respiration and intracellular ATP. However, NO is also an important regulator and mediator of numerous physiological processes in a variety of cell and tissue types that is synthesized from l-arginine by NO synthases. In an effort to determine the relative NO contributions from these competing pathways, we measured NO levels in whole cells and subcellular fractions, with and without exposure to red light, using DAF-FM, a fluorescent dye that stoichiometrically reacts with NO. Red light induced a small, but consistently reproducible, increase in fluorescence intensity in whole cells and some subcellular fractions. Whole cells exhibited the highest overall fluorescence intensity followed by (in order) cytosolic proteins, microsomes, then nuclei and mitochondria.

Cell fusion phenomena detected after in utero transplantation of Ds-red-harboring porcine amniotic fluid stem cells into EGFP transgenic mice.

PubMed

Peng, Shao-Yu; Chen, Yu-Hsu; Chou, Chih-Jen; Wang, Yao-Horng; Lee, Hung-Maan; Cheng, Winston Teng-Kui; Shaw, S W Steven; Wu, Shinn-Chih

2014-05-01

Amniotic fluid stem cells (AFSCs) are derived from the amniotic fluid of the developing fetus and can give rise to diverse differentiated cells of ectoderm, mesoderm, and endoderm lineages. Intrauterine transplantation is an approach used to cure inherited genetic fetal defects during the gestation period of pregnant dams. Certain disease such as osteogenesis imperfecta was successfully treated in affected fetal mice using this method. However, the donor cell destiny remains uncertain. The purpose of this study was to investigate the biodistribution and cell fate of Ds-red-harboring porcine AFSCs (Ds-red pAFSCs) after intrauterine transplantation into enhanced green fluorescent protein-harboring fetuses of pregnant mice. Pregnant mice (12.5 days) underwent open laparotomy with intrauterine pAFSC transplantation (5 × 10(4) cells per pup) into fetal peritoneal cavity. Three weeks after birth, the mice were sacrificed. Several samples from different organs were obtained for histological examination and flow cytometric analysis. Ds-red pAFSCs migrated most frequently into the intestines. Furthermore, enhanced green fluorescent protein and red fluorescent protein signals were co-expressed in the intestine and liver cells via immunohistochemistry studies. In utero xenotransplantation of pAFSCs fused with recipient intestinal cells instead of differentiating or maintaining the undifferentiated status in the tissue. © 2014 John Wiley & Sons, Ltd.

A FRET-facilitated photoswitching using an orange fluorescent protein with the fast photoconversion kinetics.

PubMed

Subach, Oksana M; Entenberg, David; Condeelis, John S; Verkhusha, Vladislav V

2012-09-12

Fluorescent proteins photoswitchable with noncytotoxic light irradiation and spectrally distinct from multiple available photoconvertible green-to-red probes are in high demand. We have developed a monomeric fluorescent protein, called PSmOrange2, which is photoswitchable with blue light from an orange (ex./em. at 546 nm/561 nm) to a far-red (ex./em. at 619 nm/651 nm) form. Compared to another orange-to-far-red photoconvertable variant, PSmOrange2 has blue-shifted photoswitching action spectrum, 9-fold higher photoconversion contrast, and up to 10-fold faster photoswitching kinetics. This results in the 4-fold more PSmOrange2 molecules being photoconverted in mammalian cells. Compared to common orange fluorescent proteins, such as mOrange, the orange form of PSmOrange has substantially higher photostability allowing its use in multicolor imaging applications to track dynamics of multiple populations of intracellular objects. The PSmOrange2 photochemical properties allow its efficient photoswitching with common two-photon lasers and, moreover, via Förster resonance energy transfer (FRET) from green fluorescent donors. We have termed the latter effect a FRET-facilitated photoswitching and demonstrated it using several sets of interacting proteins. The enhanced photoswitching properties of PSmOrange2 make it a superior photoconvertable protein tag for flow cytometry, conventional microscopy, and two-photon imaging of live cells.

Transgenic-cloned pigs systemically expressing red fluorescent protein, Kusabira-Orange.

PubMed

Matsunari, Hitomi; Onodera, Masafumi; Tada, Norihiro; Mochizuki, Hideki; Karasawa, Satoshi; Haruyama, Erika; Nakayama, Naoki; Saito, Hitoshi; Ueno, Satoshi; Kurome, Mayuko; Miyawaki, Atsushi; Nagashima, Hiroshi

2008-09-01

Genetically engineered pigs with cell markers such as fluorescent proteins are highly useful in lines of research that include the tracking of transplanted cells or tissues. In this study, we produced transgenic-cloned pigs carrying a gene for the newly developed red fluorescent protein, humanized Kusabira-Orange (huKO), which was cloned from the coral stone Fungia concinna. The nuclear transfer embryos, reconstructed with fetal fibroblast cells that had been transduced with huKO cDNA using retroviral vector D Delta Nsap, developed efficiently in vitro into blastocysts (28.0%, 37/132). Nearly all (94.6%, 35/37) of the cloned blastocysts derived from the transduced cells exhibited clear huKO gene expression. A total of 429 nuclear transfer embryos were transferred to four recipients, all of which became pregnant and gave birth to 18 transgenic-cloned offspring in total. All of the pigs highly expressed huKO fluorescence in all of the 23 organs and tissues analyzed, including the brain, eyes, intestinal and reproductive organs, skeletal muscle, bone, skin, and hoof. Furthermore, such expression was also confirmed by histological analyses of various tissues such as pancreatic islets, renal corpuscles, neuronal and glial cells, the retina, chondrocytes, and hematopoietic cells. These data demonstrate that transgenic-cloned pigs exhibiting systemic red fluorescence expression can be efficiently produced by nuclear transfer of somatic cells retrovirally transduced with huKO gene.

Red fluorescent protein eqFP611 and its genetically engineered dimeric variants.

PubMed

Wiedenmann, Jörg; Vallone, Beatrice; Renzi, Fabiana; Nienhaus, Karin; Ivanchenko, Sergey; Röcker, Carlheinz; Nienhaus, G Ulrich

2005-01-01

The red fluorescent protein (FP) eqFP611 from the sea anemone Entacmaea quadricolor shows favorable properties for applications as a molecular marker. Like other anthozoan FPs, it forms tetramers at physiological concentrations. The interactions among the monomers, however, are comparatively weak, as inferred from the dissociation into monomers in the presence of sodium dodecyl sulfate (SDS) or at high dilution. Analysis at the single-molecule level revealed that the monomers are highly fluorescent. For application as fusion markers, monomeric FPs are highly desirable. Therefore, we examine the monomer interfaces in the x-ray structure of eqFP611 to provide a basis for the rational design of monomeric variants. The arrangement of the four beta cans is very similar to that of other green fluorescent protein (GFP-like) proteins such as DsRed and RTMS5. A variety of structural features of the tetrameric interfaces explain the weak subunit interactions in eqFP611. We produce functional dimeric variants by introducing single point mutations in the A/B interface (Thr122Arg, Val124Thr). By contrast, structural manipulations in the A/C interface result in essentially complete loss of fluorescence, suggesting that A/C interfacial interactions play a crucial role in the folding of eqFP611 into its functional form. Copyright 2005 Society of Photo-Optical Instrumentation Engineers

Inkjet printed fluorescent nanorod layers exhibit superior optical performance over quantum dots

NASA Astrophysics Data System (ADS)

Halivni, Shira; Shemesh, Shay; Waiskopf, Nir; Vinetsky, Yelena; Magdassi, Shlomo; Banin, Uri

2015-11-01

Semiconductor nanocrystals exhibit unique fluorescence properties which are tunable in size, shape and composition. The high quantum yield and enhanced stability have led to their use in biomedical imaging and flat panel displays. Here, semiconductor nanorod based inkjet inks are presented, overcoming limitations of the commonly reported quantum dots in printing applications. Fluorescent seeded nanorods were found to be outstanding candidates for fluorescent inks, due to their low particle-particle interactions and negligible self-absorption. This is manifested by insignificant emission shifts upon printing, even in highly concentrated printed layers and by maintenance of a high fluorescence quantum yield, unlike quantum dots which exhibit fluorescence wavelength shifts and quenching effects. This behavior results from the reduced absorption/emission overlap, accompanied by low energy transfer efficiencies between the nanorods as supported by steady state and time resolved fluorescence measurements. The new seeded nanorod inks enable patterning of thin fluorescent layers, for demanding light emission applications such as signage and displays.Semiconductor nanocrystals exhibit unique fluorescence properties which are tunable in size, shape and composition. The high quantum yield and enhanced stability have led to their use in biomedical imaging and flat panel displays. Here, semiconductor nanorod based inkjet inks are presented, overcoming limitations of the commonly reported quantum dots in printing applications. Fluorescent seeded nanorods were found to be outstanding candidates for fluorescent inks, due to their low particle-particle interactions and negligible self-absorption. This is manifested by insignificant emission shifts upon printing, even in highly concentrated printed layers and by maintenance of a high fluorescence quantum yield, unlike quantum dots which exhibit fluorescence wavelength shifts and quenching effects. This behavior results from the reduced absorption/emission overlap, accompanied by low energy transfer efficiencies between the nanorods as supported by steady state and time resolved fluorescence measurements. The new seeded nanorod inks enable patterning of thin fluorescent layers, for demanding light emission applications such as signage and displays. Electronic supplementary information (ESI) available. See DOI: 10.1039/c5nr06248a

Quantum dot-engineered M13 virus layer-by-layer composite films for highly selective and sensitive turn-on TNT sensors.

PubMed

Jin, Ho; Won, Nayoun; Ahn, Boeun; Kwag, Jungheon; Heo, Kwang; Oh, Jin-Woo; Sun, Yintao; Cho, Soo Gyeong; Lee, Seung-Wuk; Kim, Sungjee

2013-07-11

We developed quantum dot-engineered M13 virus layer-by-layer hybrid composite films with incorporated fluorescence quenchers. TNT is designed to displace the quenchers and turn on the quantum dot fluorescence. TNT was detected at the sub ppb level with a high selectivity.

Direct imaging of APP proteolysis in living cells.

PubMed

Parenti, Niccoló; Del Grosso, Ambra; Antoni, Claudia; Cecchini, Marco; Corradetti, Renato; Pavone, Francesco S; Calamai, Martino

2017-01-01

Alzheimer's disease is a multifactorial disorder caused by the interaction of genetic, epigenetic and environmental factors. The formation of cytotoxic oligomers consisting of A β peptide is widely accepted as being one of the main key events triggering the development of Alzheimer's disease. A β peptide production results from the specific proteolytic processing of the amyloid precursor protein (APP). Deciphering the factors governing the activity of the secretases responsible for the cleavage of APP is still a critical issue. Kits available commercially measure the enzymatic activity of the secretases from cells lysates, in vitro . By contrast, we have developed a prototypal rapid bioassay that provides visible information on the proteolytic processing of APP directly in living cells. APP was fused to a monomeric variant of the green fluorescent protein and a monomeric variant of the red fluorescent protein at the C-terminal and N-terminal (mChAPPmGFP), respectively. Changes in the proteolytic processing rate in transfected human neuroblastoma and rat neuronal cells were imaged with confocal microscopy as changes in the red/green fluorescence intensity ratio. The significant decrease in the mean red/green ratio observed in cells over-expressing the β -secretase BACE1, or the α -secretase ADAM10, fused to a monomeric blue fluorescent protein confirms that the proteolytic site is still accessible. Specific siRNA was used to evaluate the contribution of endogenous BACE1. Interestingly, we found that the degree of proteolytic processing of APP is not completely homogeneous within the same single cell, and that there is a high degree of variability between cells of the same type. We were also able to follow with a fluorescence spectrometer the changes in the red emission intensity of the extracellular medium when BACE1 was overexpressed. This represents a complementary approach to fluorescence microscopy for rapidly detecting changes in the proteolytic processing of APP in real time. In order to allow the discrimination between the α - and the β -secretase activity, we have created a variant of mChAPPmGFP with a mutation that inhibits the α -secretase cleavage without perturbing the β -secretase processing. Moreover, we obtained a quantitatively robust estimate of the changes in the red/green ratio for the above conditions by using a flow cytometer able to simultaneously excite and measure the red and green fluorescence. Our novel approach lay the foundation for a bioassay suitable to study the effect of drugs or particular conditions, to investigate in an unbiased way the the proteolytic processing of APP in single living cells in order, and to elucidate the causes of the variability and the factors driving the processing of APP.

Ultrathin nondoped emissive layers for efficient and simple monochrome and white organic light-emitting diodes.

PubMed

Zhao, Yongbiao; Chen, Jiangshan; Ma, Dongge

2013-02-01

In this paper, highly efficient and simple monochrome blue, green, orange, and red organic light emitting diodes (OLEDs) based on ultrathin nondoped emissive layers (EMLs) have been reported. The ultrathin nondoped EML was constructed by introducing a 0.1 nm thin layer of pure phosphorescent dyes between a hole transporting layer and an electron transporting layer. The maximum external quantum efficiencies (EQEs) reached 17.1%, 20.9%, 17.3%, and 19.2% for blue, green, orange, and red monochrome OLEDs, respectively, indicating the universality of the ultrathin nondoped EML for most phosphorescent dyes. On the basis of this, simple white OLED structures are also demonstrated. The demonstrated complementary blue/orange, three primary blue/green/red, and four color blue/green/orange/red white OLEDs show high efficiency and good white emission, indicating the advantage of ultrathin nondoped EMLs on constructing simple and efficient white OLEDs.

Tuning the light emission of novel donor-acceptor phenoxazine dye-based materials towards the red spectral range

NASA Astrophysics Data System (ADS)

Damaceanu, Mariana-Dana; Constantin, Catalin-Paul

2018-04-01

A novel red fluorescent push-pull system able to generate an intramolecular charge-transfer (ICT) complex was synthesized. The novel dye (R-POX) combines some structural features which are rarely encountered in the design of other push-pull systems: hexyl-substituted phenoxazine as donor moiety, divinylketone as π-linker, and p-fluorobenzene as electron acceptor group. The relationship between the structural motif, photo-physical and electrochemical properties by UV-Vis absorption, photoluminescence and cyclic voltammetry was thoroughly investigated both as red dopant in poly(methylmethacrylate) (PMMA) or polyimide (PI) matrix, and non-doped host emitter. The molecular rigid cores of the synthesized dye formed supramolecular rod-like structures in condensed phase with a strong impact on the emissive centers. The aggregation was totally suppressed when the dye was used as dopant in an amorphous polymeric matrix, such as PMMA or PI. Electrochemical measurements revealed the dye ability for both hole and electron injection and transport. The fluorescence emission was found to be highly sensitive to solvent polarity, rendering blue-green, yellow, orange and red light emission in different organic solvents. The absolute fluorescence quantum yield reached 39.57% in solution, and dropped to 1.2% in solid state and to 14.01% when the dye was used as dopant in PMMA matrix. According to the available CIE 1931 standard, R-POX emitted pure and saturated red light of single wavelength with chromaticity coordinates very close to those of National Television System Committee (NTSC) standard red colour. The R-POX photo-optical features were compared to those of the commercial red emitter 6, 13-diphenylpentacene.

New Frontiers in NanoBiotechnology: Monitoring the Protein Function With Single Protein Resolution

DTIC Science & Technology

2005-03-29

Protein (GFP) is a spontaneously fluorescent polypeptide of 27 kD from the jellyfish Aequorea victoria that absorbs UV-blue light and emits in the...will have vast applications in science. Relationship between structure and optical properties in Green Fluorescent Proteins : A quantum mechanical study...RESEARCH AND DEVELOPMENT Invited talks Folding, stability and fluorescence efficiency of the Green and Red Fluorescent Proteins Saverio Alberti Lab.

Determination of the activity of telomerase in cancer cells by using BSA-protected gold nanoclusters as a fluorescent probe.

PubMed

Xu, Yujuan; Zhang, Peng; Wang, Zhen; Lv, Shaoping; Ding, Caifeng

2018-02-27

Gold nanoclusters (AuNCs) protected with a bovine serum albumin (BSA) coating are known to emit red fluorescence (peaking at 650 nm) on photoexcitation with ultraviolet light (365 nm). On addition of Cu(II) ions, fluorescence is quenched because Cu(II) complexes certain amino acid units in the BSA chain. Fluorescence is, however, restored if pyrophosphate (PPi) is added because it will chelate Cu(II) and remove it from the BSA coating on the AuNCs. Because PPi is involved in the function of telomerase, the BSA@AuNCs loaded with Cu(II) can act as a fluorescent probe for determination of the activity of telomerase. A fluorescent assay was worked out for telomerase that is highly sensitive and has a wide linear range (10 nU to 10 fM per mL). The fluorescent probe was applied to the determination of telomerase activity in cervix carcinoma cells via imaging. It is shown that tumor cells can be well distinguished from normal cells by monitoring the differences in intracellular telomerase activity. Graphical abstract Gold nanoclusters (AuNCs) protected by bovine serum albumin (BSA) and displaying red photoluminescence were prepared as fluorescent probe for the determination of telomerase activity and used for imaging of cervix carcinoma (HeLa) cells.

Use of image analysis to estimate anthocyanin and UV-excited fluorescent phenolic compound levels in strawberry fruit

PubMed Central

Yoshioka, Yosuke; Nakayama, Masayoshi; Noguchi, Yuji; Horie, Hideki

2013-01-01

Strawberry is rich in anthocyanins, which are responsible for the red color, and contains several colorless phenolic compounds. Among the colorless phenolic compounds, some, such as hydroxycinammic acid derivatives, emit blue-green fluorescence when excited with ultraviolet (UV) light. Here, we investigated the effectiveness of image analyses for estimating the levels of anthocyanins and UV-excited fluorescent phenolic compounds in fruit. The fruit skin and cut surface of 12 cultivars were photographed under visible and UV light conditions; colors were evaluated based on the color components of images. The levels of anthocyanins and UV-excited fluorescent compounds in each fruit were also evaluated by spectrophotometric and high performance liquid chromatography (HPLC) analyses, respectively and relationships between these levels and the image data were investigated. Red depth of the fruits differed greatly among the cultivars and anthocyanin content was well estimated based on the color values of the cut surface images. Strong UV-excited fluorescence was observed on the cut surfaces of several cultivars, and the grayscale values of the UV-excited fluorescence images were markedly correlated with the levels of those fluorescent compounds as evaluated by HPLC analysis. These results indicate that image analyses can select promising genotypes rich in anthocyanins and fluorescent phenolic compounds. PMID:23853516

Imaging fluorescent nanoparticles to probe photoinduced charging of a semiconductor-solution interface.

PubMed

Peterson, Eric M; Harris, Joel M

2013-09-24

Optically transparent semiconductors allow simultaneous control of interfacial electrical potential and spectroscopic observation of chemistry near the electrode surface. Care must be taken, however, to avoid unwanted photoexcitation-induced charging of the semiconductor electrode that could influence the results. In this work, we investigate the in situ surface charging by photoexcitation well below the band gap of an optically transparent semiconductor, indium-tin oxide (ITO) electrode. Using total-internal-reflection fluorescence microscopy, the population of ~100-nm negatively charged carboxylate-polystyrene fluorescent nanoparticles at an ITO-aqueous solution interface could be monitored in situ. At positive applied potentials (~0.7 V versus Ag/AgCl), nanoparticles accumulate reversibly in the electrical double-layer of the ITO surface, and the interfacial nanoparticle populations increase with 488-nm excitation intensity. The potential sensitivity of nanoparticle population exhibited no dependence on excitation intensity, varied from 0.1 to 10 W cm(-2), while the onset potential for particle accumulation shifted by as much as 0.3 V. This shift in surface potential appears to be due to photoexcitation-induced charging of the ITO, even though the excitation radiation photon energy, ~2.4 eV, is well below the primary band gap of ITO, >3.5 eV. A kinetic model was developed to determine the photon order of electron-hole generation relative to the electron-hole recombination. The photoexcitation process was found to be first-order in photon flux, suggesting one-photon excitation of an indirect band gap or defect sites, rather than two-photon excitation into the direct band gap. A control experiment was conducted with red-fluorescent carboxylate-polystyrene particles that were counted using 647-nm excitation, where the photon energy is below the indirect band gap or defect site energy and where the optical absorption of the film vanishes. Red illumination between 1 and 15 W cm(-2) produced no detectable shifts in the onset accumulation potential, which is consistent with the negligible optical absorption of the ITO film at this longer wavelength.

Enhanced red fluorescence in Sr2Si1-xGexO4:Eu3+ phosphors by the substitution of Si by Ge for white light emitting diodes

NASA Astrophysics Data System (ADS)

Huang, Lihui; Xu, Shiqing; Guo, Meiquan; Wang, Chenyue; Hua, Youjie; Zhao, Shilong; Deng, Degang; Wang, Huanping; Jia, Guohua

2012-07-01

Eu3+-doped Sr2Si1-xGexO4 (x=0-1) phosphors have been prepared by the high temperature solid-state reaction method. The luminescent properties of these phosphors were investigated. Red fluorescence of Eu3+ is enhanced gradually in the samples with increasing substitution of Si by Ge upon the excitation of 393 nm light. The intensity is increased by 50% with full substitution of Si by Ge. These results are originated from the structural changes and the phonon energy reduction in the samples due to the substitution of Si by Ge. The CIE chromaticity coordinates of the phosphors vary slightly around (0.62, 0.37) and all are in the red color region. The results indicate that these phosphors could be promising red phosphors for white light emitting diodes.

Fluorescence quenching and the "ring-mode" to "red-mode" transition in alkali inductively coupled plasmas

NASA Astrophysics Data System (ADS)

Huang, M.; Bazurto, R.; Camparo, J.

2018-01-01

The ring-mode to red-mode transition in alkali metal inductively coupled plasmas (ICPs) (i.e., rf-discharge lamps) is perhaps the most important physical phenomenon affecting these devices as optical pumping light sources for atomic clocks and magnetometers. It sets the limit on useful ICP operating temperature, thereby setting a limit on ICP light output for atomic-clock/magnetometer signal generation, and it is a temperature region of ICP operation associated with discharge instability. Previous work has suggested that the mechanism driving the ring-mode to red-mode transition is associated with radiation trapping, but definitive experimental evidence validating that hypothesis has been lacking. Based on that hypothesis, one would predict that the introduction of an alkali-fluorescence quenching gas (i.e., N2) into the ICP would increase the ring-mode to red-mode transition temperature. Here, we test that prediction, finding direct evidence supporting the radiation-trapping hypothesis.

Improving the photostability of bright monomeric orange and red fluorescent proteins.

PubMed

Shaner, Nathan C; Lin, Michael Z; McKeown, Michael R; Steinbach, Paul A; Hazelwood, Kristin L; Davidson, Michael W; Tsien, Roger Y

2008-06-01

All organic fluorophores undergo irreversible photobleaching during prolonged illumination. Although fluorescent proteins typically bleach at a substantially slower rate than many small-molecule dyes, in many cases the lack of sufficient photostability remains an important limiting factor for experiments requiring large numbers of images of single cells. Screening methods focusing solely on brightness or wavelength are highly effective in optimizing both properties, but the absence of selective pressure for photostability in such screens leads to unpredictable photobleaching behavior in the resulting fluorescent proteins. Here we describe an assay for screening libraries of fluorescent proteins for enhanced photostability. With this assay, we developed highly photostable variants of mOrange (a wavelength-shifted monomeric derivative of DsRed from Discosoma sp.) and TagRFP (a monomeric derivative of eqFP578 from Entacmaea quadricolor) that maintain most of the beneficial qualities of the original proteins and perform as reliably as Aequorea victoria GFP derivatives in fusion constructs.

In vivo excitation of nanoparticles using luminescent bacteria

PubMed Central

Dragavon, Joe; Blazquez, Samantha; Rekiki, Abdessalem; Samson, Chelsea; Theodorou, Ioanna; Rogers, Kelly L.; Tournebize, Régis; Shorte, Spencer L.

2012-01-01

The lux operon derived from Photorhabdus luminescens incorporated into bacterial genomes, elicits the production of biological chemiluminescence typically centered on 490 nm. The light-producing bacteria are widely used for in vivo bioluminescence imaging. However, in living samples, a common difficulty is the presence of blue-green absorbers such as hemoglobin. Here we report a characterization of fluorescence by unbound excitation from luminescence, a phenomenon that exploits radiating luminescence to excite nearby fluorophores by epifluorescence. We show that photons from bioluminescent bacteria radiate over mesoscopic distances and induce a red-shifted fluorescent emission from appropriate fluorophores in a manner distinct from bioluminescence resonance energy transfer. Our results characterizing fluorescence by unbound excitation from luminescence, both in vitro and in vivo, demonstrate how the resulting blue-to-red wavelength shift is both necessary and sufficient to yield contrast enhancement revealing mesoscopic proximity of luminescent and fluorescent probes in the context of living biological tissues. PMID:22615349

Enhanced emission of nile red fluorescent nanoparticles embedded in hybrid sol-gel glasses.

PubMed

Ferrer, Maria L; del Monte, Francisco

2005-01-13

Highly fluorescent Nile Red (NR) nanoparticles embedded in a hybrid sol-gel glass are reported. The crystallite growth within the confined system created by the porous hybrid matrix results in NR nanoparticles of averaged dimensions below 36 nm. The preparation process allows for the control of both the conformation adopted by single NR molecules prior to aggregation (e.g., near planar) and the configuration of the aggregates (e.g., oblique with phi < 54.7 degrees) prior to their assembly in the supramolecular architecture which ultimately forms the nanoparticles. The full preservation of the fluorescent configuration of the aggregates in the nanoparticles is confirmed through the application of the exciton theory, and it is responsible for the significant increase of the fluorescence emission intensity (e.g., up to 525- and 70-fold as compared to that obtained for single NR molecules embedded in pure and hybrid silica glasses, respectively).

Monitoring thioredoxin redox with a genetically encoded red fluorescent biosensor.

PubMed

Fan, Yichong; Makar, Merna; Wang, Michael X; Ai, Hui-Wang

2017-09-01

Thioredoxin (Trx) is one of the two major thiol antioxidants, playing essential roles in redox homeostasis and signaling. Despite its importance, there is a lack of methods for monitoring Trx redox dynamics in live cells, hindering a better understanding of physiological and pathological roles of the Trx redox system. In this work, we developed the first genetically encoded fluorescent biosensor for Trx redox by engineering a redox relay between the active-site cysteines of human Trx1 and rxRFP1, a redox-sensitive red fluorescent protein. We used the resultant biosensor-TrxRFP1-to selectively monitor perturbations of Trx redox in various mammalian cell lines. We subcellularly localized TrxRFP1 to image compartmentalized Trx redox changes. We further combined TrxRFP1 with a green fluorescent Grx1-roGFP2 biosensor to simultaneously monitor Trx and glutathione redox dynamics in live cells in response to chemical and physiologically relevant stimuli.

Electrosprayed synthesis of red-blood-cell-like particles with dual modality for magnetic resonance and fluorescence imaging.

PubMed

Hayashi, Koichiro; Ono, Kenji; Suzuki, Hiromi; Sawada, Makoto; Moriya, Makoto; Sakamoto, Wataru; Yogo, Toshinobu

2010-11-05

Red blood cells (RBCs) are able to avoid filtration in the spleen to prolong their half-time in the body because of their flexibility and unique shape, or a concave disk with diameter of some 10 μm. In addition, they can flow through capillary blood vessels, which are smaller than the diameter of RBCs, by morphing into a parachute-like shape. In this study, flexible RBC-like polymer particles are synthesized by electrospraying based on electrospinning. Furthermore, magnetite nanoparticles and fluorescent dye are encapsulated in the particles via in situ hydrolysis of an iron-organic compound in the presence of celluloses. The superparamagnetic behavior of the particles is confirmed by low-temperature magnetic measurements. The particles exhibited not only a dark contrast in magnetic resonance imaging (MRI), but also effective fluorescence. The RBC-like particles with flexibility are demonstrated to have a dual-modality for MRI and fluorescence imaging.

Color-coding cancer and stromal cells with genetic reporters in a patient-derived orthotopic xenograft (PDOX) model of pancreatic cancer enhances fluorescence-guided surgery

PubMed Central

Yano, Shuya; Hiroshima, Yukihiko; Maawy, Ali; Kishimoto, Hiroyuki; Suetsugu, Atsushi; Miwa, Shinji; Toneri, Makoto; Yamamoto, Mako; Katz, Matthew H.G.; Fleming, Jason B.; Urata, Yasuo; Tazawa, Hiroshi; Kagawa, Shunsuke; Bouvet, Michael; Fujiwara, Toshiyoshi; Hoffman, Robert M.

2015-01-01

Precise fluorescence-guided surgery (FGS) for pancreatic cancer has the potential to greatly improve the outcome in this recalcitrant disease. In order to achieve this goal, we have used genetic reporters to color code cancer and stroma cells in a patient-derived orthotopic xenograft (PDOX) model. The telomerase-dependent green fluorescent protein (GFP) containing adenovirus OBP401 was used to label the cancer cells of the pancreatic cancer PDOX. The PDOX was previously grown in a red fluorescent protein (RFP) transgenic mouse that stably labeled the PDOX stroma cells bright red. The color-coded PDOX model enabled FGS to completely resect the pancreatic tumors including stroma. Dual-colored FGS significantly prevented local recurrence, which bright-light surgery (BLS) or single color could not. FGS, with color-coded cancer and stroma cells has important potential for improving the outcome of recalcitrant cancer. PMID:26088297

Foliar Reflectance and Fluorescence Responses for Corn and Soybean Plants Under Nitrogen Stress

NASA Technical Reports Server (NTRS)

Middleton, E. M.; Campbell, P. K. Entcheva; Corp, L. A.; Butcher, L. M.; McMurtrey, J. E.

2003-01-01

We are investigating the use of spectral indices derived from actively induced fluorescence spectra and passive optical spectra. We examined the influence of photosynthetic pigment, carbon (C) and nitrogen (N) content on the spectral fluorescence and passive optical property characteristics of mature, upper leaves from plants provided different N fertilizer application rates: 20%, 50%, 100% and 150% of recommended N levels. A suite of optical, fluorescence, and biophysical measurements were collected on leaves from field grown corn (Zea mays L.) and soybean plants (Glycine max L.) grown in pots (greenhouse + ambient sunlight. Steady state laser-induced fluorescence emission spectra (5 nm resolution) were obtained from adaxial and abaxial surfaces resulting from excitation at single wavelengths (280, 380 or 360, and 532 nm). For emission spectra produced by each of these excitation wavelengths, ratios of emission peaks were calculated, including the red far-red chlorophyll fluorescence (ChlF) ratio (F685/F740) and the far-red/green (F740/F525) ratio. High resolution (< 3 nm) optical spectra (350-2500 nm) of reflectance, transmittance, and absorptance were also acquired for both adaxial and abaxial leaf surfaces. Species differences were demonstrated for several optical parameters. A 'red edge' derivative ratio determined from transmittance spectra [as the maximum first deivative, between 650-750 nm, normalized to the value at 744 nm, or Dmax/D744], was strongly associated with the C/N ratio (r(exp 2) = 0.90, P +/- 0.001). This ratio, calculated from reflectance spectra, was inversely related to chlorophyll b content (r(exp 2) = 0.91, P +/- 0.001) as was the ChlF (F685/F740) ratio obtained with 532 nm excitation (r(exp 2) = 0.76, P +/- 0.01). Discrimination of N treatment groups was possible with specific fluorescence band ratios (e.g., F740/F525 obtained with 380 nm excitation). Higher ChlF and blue-green emissions were measured from the abaxial leaf surfaces. Abaxial surfaces also produced higher reflectances, in general, in the 400-800 nm spectrum.

Fluorescence from polystyrene - Photochemical processes in polymeric systems, 7

NASA Technical Reports Server (NTRS)

Gupta, M. C.; Gupta, A.

1983-01-01

Results are presented for measurements of the fluorescence spectra of polystyrene in dilute solution and in pure solid films. It is determined that a major potential source of experimental error is the concurrent photooxidative degradation in air which may obscure fluorescence emission from monomeric sites in solid films at 25 C. The fluorescence spectra of oriented films are evaluated in terms of the monomer to excimer fluorescence intensity ratio and the excimer 'red shift'. The monomer to excimer fluorescence intensity ratio is determined to be significantly higher in fluid solution than in solid film.

Labeling the oily core of nanocapsules and lipid-core nanocapsules with a triglyceride conjugated to a fluorescent dye as a strategy to particle tracking in biological studies

NASA Astrophysics Data System (ADS)

Fiel, Luana Almeida; Contri, Renata Vidor; Bica, Juliane Freitas; Figueiró, Fabrício; Battastini, Ana Maria Oliveira; Guterres, Sílvia Stanisçuaski; Pohlmann, Adriana Raffin

2014-05-01

The synthesis of novel fluorescent materials represents a very important step to obtain labeled nanoformulations in order to evaluate their biological behavior. The strategy of conjugating a fluorescent dye with triacylglycerol allows that either particles differing regarding supramolecular structure, i.e., nanoemulsions, nanocapsules, lipid-core nanocapsules, or surface charge, i.e., cationic nanocapsules and anionic nanocapsules, can be tracked using the same labeled material. In this way, a rhodamine B-conjugated triglyceride was obtained to prepare fluorescent polymeric nanocapsules. Different formulations were obtained, nanocapsules (NC) or lipid-core nanocapsules (LNC), using the labeled oil and Eudragit RS100, Eudragit S100, or poly(caprolactone) (PCL), respectively. The rhodamine B was coupled with the ricinolein by activating the carboxylic function using a carbodiimide derivative. Thin layer chromatography, proton nuclear magnetic resonance (1H-NMR), Fourier transform infrared spectroscopy (FTIR), UV-vis, and fluorescence spectroscopy were used to identify the new product. Fluorescent nanocapsule aqueous suspensions were prepared by the solvent displacement method. Their pH values were 4.6 (NC-RS100), 3.5 (NC-S100), and 5.0 (LNC-PCL). The volume-weighted mean diameter ( D 4.3) and polydispersity values were 150 nm and 1.05 (NC-RS100), 350 nm and 2.28 (NC-S100), and 270 nm and 1.67 (LNC-PCL). The mean diameters determined by photon correlation spectroscopy (PCS) ( z-average) were around 200 nm. The zeta potential values were +5.85 mV (NC-RS100), -21.12 mV (NC-S100), and -19.25 mV (LNC-PCL). The wavelengths of maximum fluorescence emission were 567 nm (NC-RS100 and LNC-PCL) and 574 nm (NC-S100). Fluorescence microscopy was used to evaluate the cell uptake (human macrophage cell line) of the fluorescent nanocapsules in order to show the applicability of the approach. When the cells were treated with the fluorescent nanocapsules, red emission was detected around the cell nucleus. We demonstrated that the rhodamine B-conjugated triglyceride is a promising new material to obtain versatile dye-labeled nanocarriers presenting different chemical nature in their surfaces.

Cermet based metamaterials for multi band absorbers over NIR to LWIR frequencies

NASA Astrophysics Data System (ADS)

Pradhan, Jitendra K.; Behera, Gangadhar; Agarwal, Amit K.; Ghosh, Amitava; Ramakrishna, S. Anantha

2017-06-01

Cermets or ceramic-metals are known for their use in solar thermal technologies for their absorption across the solar band. Use of cermet layers in a metamaterial perfect absorber allows for flexible control of infra-red absorption over the short wave infra-red, to long wave infra-red bands, while keeping the visible/near infra-red absorption properties constant. We design multilayered metamaterials consisting of a conducting ground plane, a low metal volume fraction cermet/ZnS as dielectric spacer layers, and a top structured layer of an array of circular discs of metal/high volume metal fraction cermet that give rise to specified absorption bands in the near-infra-red (NIR) frequencies, as well as any specified band at SWIR-LWIR frequencies. Thus, a complete decoupling of the absorption at optical/NIR frequencies and the infra-red absorption behaviour of a structured metamaterial is demonstrated.

Changes in the spectral properties of a plasma membrane lipid analog during the first seconds of endocytosis in living cells.

PubMed Central

Chen, C S; Martin, O C; Pagano, R E

1997-01-01

N-[5-(5, 7-dimethyl Bodipy)-1-pentanoyl]-D-erythro-sphingosylphosphorylcholine (C5-DMB-SM), a fluorescent analog of sphingomyelin, has been used in a study of the formation of very early endosomes in human skin fibroblasts. This lipid exhibits a shift in its fluorescence emission maximum from green (approximately 515 nm) to red (approximately 620 nm) wavelengths with increasing concentrations in membranes. When cells were incubated with 5 microM C5-DMB-SM at 4 degrees C and washed, only plasma membrane fluorescence (yellow-green) was observed. When these cells were briefly (< or = 1 min) warmed to 37 degrees C to allow internalization to occur, and then incubated with defatted bovine serum albumin (back-exchanged) at 11 degrees C to remove fluorescent lipids from the plasma membrane, C5-DMB-SM was distributed in a punctate pattern throughout the cytoplasm. Interestingly, within the same cell some endosomes exhibited green fluorescence, whereas others emitted red-orange fluorescence. Furthermore, the red-orange endosomes were usually seen at the periphery of the cell, while the green endosomes were more uniformly distributed throughout the cytoplasm. This mixed population of endosomes was seen after internalization times as short as 7 s and was also seen over a wide range of C5-DMB-SM concentrations (1-25 microM). Control experiments established that the variously colored endosomes were not induced by changes in pH, membrane potential, vesicle size, or temperature. Quantitative fluorescence microscopy demonstrated that the apparent concentration of the lipid analog in the red-orange endosomes was severalfold higher than its initial concentration at the plasma membrane, suggesting selective internalization (sorting) of the lipid into a subset of early endosomes. Colocalization studies using C5-DMB-SM and either anti-transferrin receptor antibodies or fluorescently labeled low-density lipoprotein further demonstrated that this subpopulation of endosomes resulted from receptor-mediated endocytosis. We conclude that the spectral properties of C5-DMB-SM can be used to distinguish unique populations of early endosomes from one another and to record dynamic changes in their number and distribution within living cells. Images FIGURE 1 FIGURE 3 FIGURE 4 FIGURE 6 FIGURE 8 FIGURE 9 PMID:8994591

Mitochondrial dynamics and optical conformation changes in DsRed as studied by Fourier imaging correlation spectroscopy

NASA Astrophysics Data System (ADS)

Senning, Eric Nicolas

Novel experiments that probe the dynamics of intracellular species, including the center-of-mass displacements and internal conformational transitions of biological macromolecules, have the potential to reveal the complex biochemical mechanisms operating within the cell. This work presents the implementation and development of Fourier imaging correlation spectroscopy (FICS), a phase-selective approach to fluorescence spectroscopy that measures the collective coordinate fluctuations of fluorescently labeled microscopic particles. In FICS experiments, a spatially modulated optical grating excites a fluorescently labeled sample. Phase-synchronous detection of the fluorescence, with respect to the phase of the exciting optical grating, can be used to monitor the fluctuations of partially averaged spatial coordinates. These data are then analyzed by two-point and four-point time correlation functions to provide a statistically meaningful understanding of the dynamics under observation. FICS represents a unique route to elevate signal levels, while acquiring detailed information about molecular coordinate trajectories. Mitochondria of mammalian cells are known to associate with cytoskeletal proteins, and their motions are affected by the stability of microtubules and microfilaments. Within the cell it is possible to fluorescently label the mitochondria and study its dynamic behavior with FICS. The dynamics of S. cerevisiae yeast mitochondria are characterized at four discrete length scales (ranging from 0.6--1.19 mum) and provide detailed information about the influence of specific cytoskeletal elements. Using the microtubule and microfilament destabilizing agents, Nocodazole and Latrunculin A, it is determined that microfilaments are required for normal yeast mitochondrial motion while microtubules have no effect. Experiments with specific actin mutants revealed that actin is responsible for enhanced mobility on length scales greater than 0.6 mum. The versatility of FICS expands when individual molecules are labeled with fluorescent chromophores. In recent experiments on the tetrameric fluorescent protein DsRed, polarization-modulated FICS (PM-FICS) is demonstrated to separate conformational dynamics from molecular translational dynamics. The optical switching pathways of DsRed, a tetrameric complex of fluorescent protein subunits, are examined. An analysis of PM-FICS coordinate trajectories, in terms of 2D spectra and joint probability distributions, provides detailed information about the transition pathways between distinct dipole-coupled DsRed conformations. This dissertation includes co-authored and previously published material.

Fluorescent solute-partitioning characterization of layered soft contact lenses.

PubMed

Dursch, T J; Liu, D E; Oh, Y; Radke, C J

2015-03-01

Partitioning of aqueous packaging, wetting, and care-solution agents into and out of soft contact lenses (SCLs) is important for improving wear comfort and also for characterizing lens physico-chemical properties. We illustrate both features of partitioning by application of fluorescent-solute partitioning into DAILIES TOTAL1® (delefilcon A) water-gradient SCLs, which exhibit a layered structure of a silicone-hydrogel (SiHy) core sandwiched between thin surface-gel layers. Two-photon fluorescence confocal laser-scanning microscopy and attenuated total-reflectance Fourier-transform infrared spectroscopy (ATR-FTIR) characterize the lens and assess uptake profiles of six prototypical fluorescent solutes. Comparison of solute uptake in a SiHy-core prototype lens (i.e., O2OPTIX(TM)) validates the core SiHy structure of DAILIESTOTAL1®. To establish surface-layer charge, partition coefficients and water contents are obtained for aqueous pH values of 4 and 7.4. Solute fluorescence-intensity profiles clearly confirm a layered structure for the DAILIES TOTAL1® lenses. In all cases, aqueous solute partition coefficients are greater in the surface layers than in the SiHy core, signifying higher water in the surface gels. ATR-FTIR confirms surface-layer mass water contents of 82±3%. Water uptake and hydrophilic-solute uptake at pH 4 compared with that at pH 7.4 reveal that the surface-gel layers are anionic at physiologic pH 7.4, whereas both the SiHy core and O2OPTIX™ (lotrafilcon B) are nonionic. We successfully confirm the layered structure of DAILIES TOTAL1®, consisting of an 80-μm-thick SiHy core surrounded by 10-μm-thick polyelectrolyte surface-gel layers of significantly greater water content and aqueous solute uptake compared with the core. Accordingly, fluorescent-solute partitioning in SCLs provides information on gel structure and composition, in addition to quantifying uptake and release amounts and rates. Copyright © 2014 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Selective labeling of a single organelle by using two-photon conversion of a photoconvertible fluorescent protein

NASA Astrophysics Data System (ADS)

Watanabe, Wataru; Shimada, Tomoko; Matsunaga, Sachihiro; Kurihara, Daisuke; Arimura, Shin-ichi; Tsutsumi, Nobuhiro; Fukui, Kiichi; Itoh, Kazuyoshi

2008-02-01

We present space-selective labeling of organelles by using two-photon conversion of a photoconvertible fluorescent protein with near-infrared femtosecond laser pulses. Two-photon excitation of photoconvertible fluorescent-protein, Kaede, enables space-selective labeling of organelles. We alter the fluorescence of target mitochondria in a tobacco BY-2 cell from green to red by focusing femtosecond laser pulses with a wavelength of 750 nm.

Improving dynamic phytoplankton reserve-utilization models with an indirect proxy for internal nitrogen.

PubMed

Malerba, Martino E; Heimann, Kirsten; Connolly, Sean R

2016-09-07

Ecologists have often used indirect proxies to represent variables that are difficult or impossible to measure directly. In phytoplankton, the internal concentration of the most limiting nutrient in a cell determines its growth rate. However, directly measuring the concentration of nutrients within cells is inaccurate, expensive, destructive, and time-consuming, substantially impairing our ability to model growth rates in nutrient-limited phytoplankton populations. The red chlorophyll autofluorescence (hereafter "red fluorescence") signal emitted by a cell is highly correlated with nitrogen quota in nitrogen-limited phytoplankton species. The aim of this study was to evaluate the reliability of including flow cytometric red fluorescence as a proxy for internal nitrogen status to model phytoplankton growth rates. To this end, we used the classic Quota model and designed three approaches to calibrate its model parameters to data: where empirical observations on cell internal nitrogen quota were used to fit the model ("Nitrogen-Quota approach"), where quota dynamics were inferred only from changes in medium nutrient depletion and population density ("Virtual-Quota approach"), or where red fluorescence emission of a cell was used as an indirect proxy for its internal nitrogen quota ("Fluorescence-Quota approach"). Two separate analyses were carried out. In the first analysis, stochastic model simulations were parameterized from published empirical relationships and used to generate dynamics of phytoplankton communities reared under nitrogen-limited conditions. Quota models were fitted to the dynamics of each simulated species with the three different approaches and the performance of each model was compared. In the second analysis, we fit Quota models to laboratory time-series and we calculate the ability of each calibration approach to describe the observed trajectories of internal nitrogen quota in the culture. Results from both analyses concluded that the Fluorescence-Quota approach including per-cell red fluorescence as a proxy of internal nitrogen substantially improved the ability of Quota models to describe phytoplankton dynamics, while still accounting for the biologically important process of cell nitrogen storage. More broadly, many population models in ecology implicitly recognize the importance of accounting for storage mechanisms to describe the dynamics of individual organisms. Hence, the approach documented here with phytoplankton dynamics may also be useful for evaluating the potential of indirect proxies in other ecological systems. Copyright © 2016 Elsevier Ltd. All rights reserved.

Photomorphogenesis, photosynthesis, and seed yield of wheat plants grown under red light-emitting diodes (LEDs) with and without supplemental blue lighting

NASA Technical Reports Server (NTRS)

Goins, G. D.; Yorio, N. C.; Sanwo, M. M.; Brown, C. S.; Sager, J. C. (Principal Investigator)

1997-01-01

Red light-emitting diodes (LEDs) are a potential light source for growing plants in spaceflight systems because of their safety, small mass and volume, wavelength specificity, and longevity. Despite these attractive features, red LEDs must satisfy requirements for plant photosynthesis and photomorphogenesis for successful growth and seed yield. To determine the influence of gallium aluminium arsenide (GaAlAs) red LEDs on wheat photomorphogenesis, photosynthesis, and seed yield, wheat (Triticum aestivum L., cv. 'USU-Super Dwarf') plants were grown under red LEDs and compared to plants grown under daylight fluorescent (white) lamps and red LEDs supplemented with either 1% or 10% blue light from blue fluorescent (BF) lamps. Compared to white light-grown plants, wheat grown under red LEDs alone demonstrated less main culm development during vegetative growth through preanthesis, while showing a longer flag leaf at 40 DAP and greater main culm length at final harvest (70 DAP). As supplemental BF light was increased with red LEDs, shoot dry matter and net leaf photosynthesis rate increased. At final harvest, wheat grown under red LEDs alone displayed fewer subtillers and a lower seed yield compared to plants grown under white light. Wheat grown under red LEDs+10% BF light had comparable shoot dry matter accumulation and seed yield relative to wheat grown under white light. These results indicate that wheat can complete its life cycle under red LEDs alone, but larger plants and greater amounts of seed are produced in the presence of red LEDs supplemented with a quantity of blue light.

The application of laser induced predissociation fluorescence to the measurement of vibrational temperatures in a shock layer flow

NASA Technical Reports Server (NTRS)

Sutton, D. J.; Houwing, A. F. P.; Palma, P. C.; Boyce, R. R.; Sandeman, R. J.; Mundt, CH.

1993-01-01

Single shot spatially and spectrally resolved laser induced predissociation fluorescence measurements in a shock layer around a cylinder in a pulsed supersonic free stream are presented. Fluorescence signals were produced using the tuned output of an argon fluoride excimer laser to excite a mixture of rovibrational transitions in molecular oxygen. The signals produced along a line inside the shock layer were focussed onto a two dimensional detector coupled to a spectrometer, thus allowing spectral and spatial resolution of the fluorescence. In this way, it was possible to detect two fluorescence signals from two different transitions simultaneously, allowing the determination of vibrational temperatures without the need for calibration. However, to minimize problems associated with low signal to noise ratios, background subtraction and spatial averaging was required. The experimental measurements are compared with theoretical inviscid shock layer calculations for nonequilibrium air. A description of the strategies employed in these calculations is also provided.

Chair-side detection of Prevotella Intermedia in mature dental plaque by its fluorescence.

PubMed

Nomura, Yoshiaki; Takeuchi, Hiroaki; Okamoto, Masaaki; Sogabe, Kaoru; Okada, Ayako; Hanada, Nobuhiro

2017-06-01

Prevotella intermedia/nigrescens is one of the well-known pathogens causing periodontal diseases, and the red florescence excited by the visible blue light caused by the protoporphyrin IX in the bacterial cells could be useful for the chair-side detection. The aim of this study was to evaluated levels of periodontal pathogen, especially P. intermedia in clinical samples of red fluorescent dental plaque. Thirty two supra gingival plaque samples from six individuals were measured its fluorescence at 640nm wavelength excited by 409nm. Periodontopathic bacteria were counted by the Invader PLUS PCR assay. Co-relations the fluorescence intensity and bacterial counts were analyzed by Person's correlation coefficient and simple and multiple regression analysis. Positive and negative predictive values of the fluorescence intensities for with or without P. intermedia in supragingival plaque was calculated. When relative fluorescence unit (RFU) were logarithmic transformed, statistically significant linear relations between RFU and bacterial counts were obtained for P. intermedia, Porphyromonas gingivalis and Tannerella forsythia. By the multiple regression analysis, only P. intermedia had statistically significant co-relation with fluorescence intensities. All of the fluorescent dental plaque contained P. intermedia m. In contrast, 28% of non-fluorescent plaques contained P. intermedia. To check the fluorescence dental plaque in the oral cavity could be the simple chair-side screening of the mature dental plaque before examining the periodontal pathogens especially P. intermedia by the PCR method. Copyright © 2017 Elsevier B.V. All rights reserved.

Color transitions in coral's fluorescent proteins by site-directed mutagenesis

PubMed Central

Gurskaya, Nadya G; Savitsky, Alexander P; Yanushevich, Yurii G; Lukyanov, Sergey A; Lukyanov, Konstantin A

2001-01-01

Background Green Fluorescent Protein (GFP) cloned from jellyfish Aequorea victoria and its homologs from corals Anthozoa have a great practical significance as in vivo markers of gene expression. Also, they are an interesting puzzle of protein science due to an unusual mechanism of chromophore formation and diversity of fluorescent colors. Fluorescent proteins can be subdivided into cyan (~ 485 nm), green (~ 505 nm), yellow (~ 540 nm), and red (>580 nm) emitters. Results Here we applied site-directed mutagenesis in order to investigate the structural background of color variety and possibility of shifting between different types of fluorescence. First, a blue-shifted mutant of cyan amFP486 was generated. Second, it was established that cyan and green emitters can be modified so as to produce an intermediate spectrum of fluorescence. Third, the relationship between green and yellow fluorescence was inspected on closely homologous green zFP506 and yellow zFP538 proteins. The following transitions of colors were performed: yellow to green; yellow to dual color (green and yellow); and green to yellow. Fourth, we generated a mutant of cyan emitter dsFP483 that demonstrated dual color (cyan and red) fluorescence. Conclusions Several amino acid substitutions were found to strongly affect fluorescence maxima. Some positions primarily found by sequence comparison were proved to be crucial for fluorescence of particular color. These results are the first step towards predicting the color of natural GFP-like proteins corresponding to newly identified cDNAs from corals. PMID:11459517

Long-wavelength (red) light produces hyperopia in juvenile and adolescent tree shrews.

PubMed

Gawne, Timothy J; Ward, Alexander H; Norton, Thomas T

2017-11-01

In infant tree shrews, exposure to narrow-band long-wavelength (red) light, that stimulates long-wavelength sensitive cones almost exclusively, slows axial elongation and produces hyperopia. We asked if red light produces hyperopia in juvenile and adolescent animals, ages when plus lenses are ineffective. Animals were raised in fluorescent colony lighting (100-300 lux) until they began 13days of red-light treatment at 11 (n=5, "infant"), 35 (n=5, "juvenile") or 95 (n=5, "adolescent") days of visual experience (DVE). LEDs provided 527-749 lux on the cage floor. To control for the higher red illuminance, a fluorescent control group (n=5) of juvenile (35 DVE) animals was exposed to ∼975 lux. Refractions were measured daily; ocular component dimensions at the start and end of treatment and end of recovery in colony lighting. These groups were compared with normals (n=7). In red light, the refractive state of both juvenile and adolescent animals became significantly (P<0.05) hyperopic: juvenile 3.9±1.0 diopters (D, mean±SEM) vs. normal 0.8±0.1D; adolescent 1.6±0.2D vs. normal 0.4±0.1D. The fluorescent control group refractions (0.6±0.3D) were normal. In red-treated juveniles the vitreous chamber was significantly smaller than normal (P<0.05): juvenile 2.67±0.03mmvs. normal 2.75±0.02mm. The choroid was also significantly thicker: juvenile 77±4μmvs. normal 57±3μm (P<0.05). Although plus lenses do not restrain eye growth in juvenile tree shrews, the red light-induced slowed growth and hyperopia in juvenile and adolescent tree shrews demonstrates that the emmetropization mechanism is still capable of restraining eye growth at these ages. Copyright © 2017 Elsevier Ltd. All rights reserved.

Diffuse fluorescence tomography of exo- and endogenously labeled tumors

NASA Astrophysics Data System (ADS)

Balalaeva, Irina V.; Turchin, Ilya V.; Orlova, Anna G.; Plekhanov, Vladimir I.; Shirmanova, Marina V.; Kleshnin, Michail S.; Fiks, Ilya I.; Zagainova, Elena V.; Kamensky, Vladislav A.

2007-06-01

Strong light scattering and absorption limit observation of the internal structure of biological tissue. Only special tools for turbid media imaging, such as optical diffuse tomography, enable noninvasive investigation of the internal biological tissues, including visualization and intravital monitoring of deep tumors. In this work the preliminary results of diffuse fluorescence tomography (DFT) of small animals are presented. Usage of exogenous fluorophores, targeted specifically at tumor cells, and fluorescent proteins expressed endogenously can significantly increase the contrast of obtained images. Fluorescent compounds of different nature, such as sulphonated aluminium phthalocyanine (Photosens), red fluorescing proteins and CdTe/CdSe-core/shell nanocrystals (quantum dots) were applied. We tested diffuse fluorescence tomography method at model media, in post mortem and in vivo experiments. The animal was scanned in transilluminative configuration by low-frequency modulated light (1 kHz) from Nd:YAG laser with second harmonic generation at wavelength of 532 nm or semiconductor laser at wavelength of 655 nm. Quantum dots or protein DsRed2 in glass capsules (inner diameter 2-3 mm) were placed post mortem inside the esophagus of 7-day-old hairless rats to simulate marked tumors. Photosens was injected intravenously to linear mice with metastazing Lewis lung carcinoma. The reconstruction algorithm, based on Algebraic Reconstruction Technique, was created and tested numerically in model experiments. High contrast images of tumor simulating capsules with DsRed2 concentrations about 10 -6 M and quantum dots about 5x10 -11 M have been obtained. Organ distribution of Photosens and its accumulation in tumors and surrounding tissues of animals has been examined. We have conducted the monitoring of tumors, exogenously labeled by photosensitizer. This work demonstrates potential capabilities of DFT method for intravital detection and monitoring of deep fluorescent-labeled tumors in animal models. The comparative analysis of conventional photosensitizer, fluorescent proteins and quantum dots has been carried out.

Space station image captures a red tide ciliate bloom at high spectral and spatial resolution.

PubMed

Dierssen, Heidi; McManus, George B; Chlus, Adam; Qiu, Dajun; Gao, Bo-Cai; Lin, Senjie

2015-12-01

Mesodinium rubrum is a globally distributed nontoxic ciliate that is known to produce intense red-colored blooms using enslaved chloroplasts from its algal prey. Although frequent enough to have been observed by Darwin, blooms of M. rubrum are notoriously difficult to quantify because M. rubrum can aggregate into massive clouds of rusty-red water in a very short time due to its high growth rates and rapid swimming behavior and can disaggregate just as quickly by vertical or horizontal dispersion. A September 2012 hyperspectral image from the Hyperspectral Imager for the Coastal Ocean sensor aboard the International Space Station captured a dense red tide of M. rubrum (10(6) cells per liter) in surface waters of western Long Island Sound. Genetic data confirmed the identity of the chloroplast as a cryptophyte that was actively photosynthesizing. Microscopy indicated extremely high abundance of its yellow fluorescing signature pigment phycoerythrin. Spectral absorption and fluorescence features were related to ancillary photosynthetic pigments unique to this organism that cannot be observed with traditional satellites. Cell abundance was estimated at a resolution of 100 m using an algorithm based on the distinctive yellow fluorescence of phycoerythrin. Future development of hyperspectral satellites will allow for better enumeration of bloom-forming coastal plankton, the associated physical mechanisms, and contributions to marine productivity.

Fluorescent transgenic zebrafish as a biosensor for growth-related effects of methyl parathion.

PubMed

Almeida, Daniela Volcan; Vaz, Bernardo; Azevedo Figueiredo, Márcio; Junior, Antonio Sergio Varela; Marins, Luis Fernando

2014-07-01

Transgenic fish models are potential alternative subjects in toxicological studies, since they can provide in vivo information on the deleterious effects of different substances. Here, we used a transgenic zebrafish (Danio rerio) lineage, which expresses a destabilized fluorescent protein (DsRED) driven by the myosin light chain promoter (Mylz2), in order to propose a new research tool for environmental biomonitoring. For validating the MYO-RED lineage, we exposed fish to the organophosphorated pesticide methyl parathion (MP). The effect of MP on fish growth was assessed by evaluating weight, length, condition factor and muscle fiber diameter. All factors suffered reduction at both tested concentrations (0.13μM and 13μM of MP). Similarly, fluorescence intensity decreased in a concentration-dependent manner, suggesting muscle protein catabolism. However, DsRED gene expression lowered only at the higher MP concentration. Results indicate that the MYO-RED transgenic zebrafish is an interesting model for detecting the growth-related effects of pollutants. Destabilized proteins such as reporter genes are apparently sensitive biomarkers, since effects were observed even at the lower, environmentally acceptable concentration. Therefore, this transgenic fish is a promising candidate model for sensitive, fast, and easy environmental monitoring. Copyright © 2014 Elsevier B.V. All rights reserved.

[Construction and expression analysis of the zebrafish heart-specific transgenetic vector based on Tol2 transposable element].

PubMed

Chen, Tingfang; Luo, Na; Xie, Huaping; Wu, Xiushan; Deng, Yun

2010-02-01

In an effort to generate a desired expression construct for making heart-specific expression transgenic zebrafish, a Tol2 plasmid, which can drive EGFP reporter gene specifically expressed in the heart, was modified using subcloning technology. An IRES fragment bearing multiple cloning site (MCS) was amplified directly from pIRES2-EGFP plasmid and was inserted between the CMLC2 promoter and EGFP fragment of the pDestTol2CG vector. This recombinant expression plasmid pTol2-CMLC2-IRES-EGFP can drive any interested gene specifically expressed in the zebrafish heart along with EGFP reporter gene. To test the effectiveness of this new expression plasmid, we constructed pTol2-CMLC2-RED-IRES-EGFP plasmid by inserting another reporter gene DsRed-Monome into MCS downstream of the CMLC2 promoter and injected this transgenic recombinant plasmid into one-cell stage embryos of zebrafish. Under fluorescence microscope, both the red fluorescence and the green fluorescence produced by pTol2-CMLC2-RED-IRES-EGFP were detected specifically in the heart tissue in the same expression pattern. This novel expression construct pTol2-CMLC2-IRES-EGFP will become an important tool for our research on identifying heart development candidate genes' function using zebrafish as a model.

Improving brightness and photostability of green and red fluorescent proteins for live cell imaging and FRET reporting.

PubMed

Bajar, Bryce T; Wang, Emily S; Lam, Amy J; Kim, Bongjae B; Jacobs, Conor L; Howe, Elizabeth S; Davidson, Michael W; Lin, Michael Z; Chu, Jun

2016-02-16

Many genetically encoded biosensors use Förster resonance energy transfer (FRET) to dynamically report biomolecular activities. While pairs of cyan and yellow fluorescent proteins (FPs) are most commonly used as FRET partner fluorophores, respectively, green and red FPs offer distinct advantages for FRET, such as greater spectral separation, less phototoxicity, and lower autofluorescence. We previously developed the green-red FRET pair Clover and mRuby2, which improves responsiveness in intramolecular FRET reporters with different designs. Here we report the engineering of brighter and more photostable variants, mClover3 and mRuby3. mClover3 improves photostability by 60% and mRuby3 by 200% over the previous generation of fluorophores. Notably, mRuby3 is also 35% brighter than mRuby2, making it both the brightest and most photostable monomeric red FP yet characterized. Furthermore, we developed a standardized methodology for assessing FP performance in mammalian cells as stand-alone markers and as FRET partners. We found that mClover3 or mRuby3 expression in mammalian cells provides the highest fluorescence signals of all jellyfish GFP or coral RFP derivatives, respectively. Finally, using mClover3 and mRuby3, we engineered an improved version of the CaMKIIα reporter Camuiα with a larger response amplitude.

Space station image captures a red tide ciliate bloom at high spectral and spatial resolution

PubMed Central

Dierssen, Heidi; McManus, George B.; Chlus, Adam; Qiu, Dajun; Gao, Bo-Cai; Lin, Senjie

2015-01-01

Mesodinium rubrum is a globally distributed nontoxic ciliate that is known to produce intense red-colored blooms using enslaved chloroplasts from its algal prey. Although frequent enough to have been observed by Darwin, blooms of M. rubrum are notoriously difficult to quantify because M. rubrum can aggregate into massive clouds of rusty-red water in a very short time due to its high growth rates and rapid swimming behavior and can disaggregate just as quickly by vertical or horizontal dispersion. A September 2012 hyperspectral image from the Hyperspectral Imager for the Coastal Ocean sensor aboard the International Space Station captured a dense red tide of M. rubrum (106 cells per liter) in surface waters of western Long Island Sound. Genetic data confirmed the identity of the chloroplast as a cryptophyte that was actively photosynthesizing. Microscopy indicated extremely high abundance of its yellow fluorescing signature pigment phycoerythrin. Spectral absorption and fluorescence features were related to ancillary photosynthetic pigments unique to this organism that cannot be observed with traditional satellites. Cell abundance was estimated at a resolution of 100 m using an algorithm based on the distinctive yellow fluorescence of phycoerythrin. Future development of hyperspectral satellites will allow for better enumeration of bloom-forming coastal plankton, the associated physical mechanisms, and contributions to marine productivity. PMID:26627232

Rational Design of a Triple Reporter Gene for Multimodality Molecular Imaging

PubMed Central

Hsieh, Ya-Ju; Ke, Chien-Chih; Yeh, Skye Hsin-Hsien; Lin, Chien-Feng; Chen, Fu-Du; Lin, Kang-Ping; Chen, Ran-Chou; Liu, Ren-Shyan

2014-01-01

Multimodality imaging using noncytotoxic triple fusion (TF) reporter genes is an important application for cell-based tracking, drug screening, and therapy. The firefly luciferase (fl), monomeric red fluorescence protein (mrfp), and truncated herpes simplex virus type 1 thymidine kinase SR39 mutant (ttksr39) were fused together to create TF reporter gene constructs with different order. The enzymatic activities of TF protein in vitro and in vivo were determined by luciferase reporter assay, H-FEAU cellular uptake experiment, bioluminescence imaging, and micropositron emission tomography (microPET). The TF construct expressed in H1299 cells possesses luciferase activity and red fluorescence. The tTKSR39 activity is preserved in TF protein and mediates high levels of H-FEAU accumulation and significant cell death from ganciclovir (GCV) prodrug activation. In living animals, the luciferase and tTKSR39 activities of TF protein have also been successfully validated by multimodality imaging systems. The red fluorescence signal is relatively weak for in vivo imaging but may expedite FACS-based selection of TF reporter expressing cells. We have developed an optimized triple fusion reporter construct DsRedm-fl-ttksr39 for more effective and sensitive in vivo animal imaging using fluorescence, bioluminescence, and PET imaging modalities, which may facilitate different fields of biomedical research and applications. PMID:24809057

High-throughput living cell-based optical biosensor for detection of bacterial lipopolysaccharide (LPS) using a red fluorescent protein reporter system.

PubMed

Jiang, Hui; Jiang, Donglei; Shao, Jingdong; Sun, Xiulan; Wang, Jiasheng

2016-11-14

Due to the high toxicity of bacterial lipopolysaccharide (LPS), resulting in sepsis and septic shock, two major causes of death worldwide, significant effort is directed toward the development of specific trace-level LPS detection systems. Here, we report sensitive, user-friendly, high-throughput LPS detection in a 96-well microplate using a transcriptional biosensor system, based on 293/hTLR4A-MD2-CD14 cells that are transformed by a red fluorescent protein (mCherry) gene under the transcriptional control of an NF-κB response element. The recognition of LPS activates the biosensor cell, TLR4, and the co-receptor-induced NF-κB signaling pathway, which results in the expression of mCherry fluorescent protein. The novel cell-based biosensor detects LPS with specificity at low concentration. The cell-based biosensor was evaluated by testing LPS isolated from 14 bacteria. Of the tested bacteria, 13 isolated Enterobacteraceous LPSs with hexa-acylated structures were found to increase red fluorescence and one penta-acylated LPS from Pseudomonadaceae appeared less potent. The proposed biosensor has potential for use in the LPS detection in foodstuff and biological products, as well as bacteria identification, assisting the control of foodborne diseases.

TSCA Environmental Release Application (TERA) for Alcaligenes xylosoxidans subspecies denitrificans strain AL6.1

EPA Pesticide Factsheets

TERA submitted by University of California, Riverside and given the tracking designation of R-03-0001. The microorganism has been modified to carry a coding sequence of DsRed for expressing a red fluorescent protein.

Directed evolution methods for improving polypeptide folding and solubility and superfolder fluorescent proteins generated thereby

DOEpatents

Waldo, Geoffrey S.

2007-09-18

The current invention provides methods of improving folding of polypeptides using a poorly folding domain as a component of a fusion protein comprising the poorly folding domain and a polypeptide of interest to be improved. The invention also provides novel green fluorescent proteins (GFPs) and red fluorescent proteins that have enhanced folding properties.

Single Cell Transfection through Precise Microinjection with Quantitatively Controlled Injection Volumes

NASA Astrophysics Data System (ADS)

Chow, Yu Ting; Chen, Shuxun; Wang, Ran; Liu, Chichi; Kong, Chi-Wing; Li, Ronald A.; Cheng, Shuk Han; Sun, Dong

2016-04-01

Cell transfection is a technique wherein foreign genetic molecules are delivered into cells. To elucidate distinct responses during cell genetic modification, methods to achieve transfection at the single-cell level are of great value. Herein, we developed an automated micropipette-based quantitative microinjection technology that can deliver precise amounts of materials into cells. The developed microinjection system achieved precise single-cell microinjection by pre-patterning cells in an array and controlling the amount of substance delivered based on injection pressure and time. The precision of the proposed injection technique was examined by comparing the fluorescence intensities of fluorescent dye droplets with a standard concentration and water droplets with a known injection amount of the dye in oil. Injection of synthetic modified mRNA (modRNA) encoding green fluorescence proteins or a cocktail of plasmids encoding green and red fluorescence proteins into human foreskin fibroblast cells demonstrated that the resulting green fluorescence intensity or green/red fluorescence intensity ratio were well correlated with the amount of genetic material injected into the cells. Single-cell transfection via the developed microinjection technique will be of particular use in cases where cell transfection is challenging and genetically modified of selected cells are desired.

Highly selective and sensitive nanoprobes for cyanide based on gold nanoclusters with red fluorescence emission

NASA Astrophysics Data System (ADS)

Zhang, Guomei; Qiao, Yunyun; Xu, Ting; Zhang, Caihong; Zhang, Yan; Shi, Lihong; Shuang, Shaomin; Dong, Chuan

2015-07-01

We report a novel and environmentally friendly fluorescent probe for detecting the cyanide ion (CN-) using l-amino acid oxidase (LAAOx)-protected Au nanoclusters (LAAOx@AuNCs) with red emission. The fluorescence-based sensing behaviour of LAAOx@AuNCs towards anions was investigated in buffered aqueous media. Among the anions studied, CN- was found to effectively quench the fluorescence emission of AuNCs based on CN- induced Au core decomposition. Excellent sensitivity and selectivity toward the detection of CN- in aqueous solution were observed. The CN- detection limit was determined to be approximately 180 nM, which is 15 times lower than the maximum level (2700 nM) of CN- in drinking water permitted by the World Health Organization (WHO). A linear relationship between the fluorescence intensity and CN- concentration was observed in two ranges of CN- concentration, including 3.2 × 10-6 to 3.4 × 10-5 mol L-1 and 3.81 × 10-5 to 1.04 × 10-4 mol L-1. The high sensitivity and selectivity to CN- among the 17 types of anions make the AuNCs good candidates for use in fluorescent nanoprobes of CN-.

Single Cell Transfection through Precise Microinjection with Quantitatively Controlled Injection Volumes.

PubMed

Chow, Yu Ting; Chen, Shuxun; Wang, Ran; Liu, Chichi; Kong, Chi-Wing; Li, Ronald A; Cheng, Shuk Han; Sun, Dong

2016-04-12

Cell transfection is a technique wherein foreign genetic molecules are delivered into cells. To elucidate distinct responses during cell genetic modification, methods to achieve transfection at the single-cell level are of great value. Herein, we developed an automated micropipette-based quantitative microinjection technology that can deliver precise amounts of materials into cells. The developed microinjection system achieved precise single-cell microinjection by pre-patterning cells in an array and controlling the amount of substance delivered based on injection pressure and time. The precision of the proposed injection technique was examined by comparing the fluorescence intensities of fluorescent dye droplets with a standard concentration and water droplets with a known injection amount of the dye in oil. Injection of synthetic modified mRNA (modRNA) encoding green fluorescence proteins or a cocktail of plasmids encoding green and red fluorescence proteins into human foreskin fibroblast cells demonstrated that the resulting green fluorescence intensity or green/red fluorescence intensity ratio were well correlated with the amount of genetic material injected into the cells. Single-cell transfection via the developed microinjection technique will be of particular use in cases where cell transfection is challenging and genetically modified of selected cells are desired.

13-fold resolution gain through turbid layer via translated unknown speckle illumination

PubMed Central

Guo, Kaikai; Zhang, Zibang; Jiang, Shaowei; Liao, Jun; Zhong, Jingang; Eldar, Yonina C.; Zheng, Guoan

2017-01-01

Fluorescence imaging through a turbid layer holds great promise for various biophotonics applications. Conventional wavefront shaping techniques aim to create and scan a focus spot through the turbid layer. Finding the correct input wavefront without direct access to the target plane remains a critical challenge. In this paper, we explore a new strategy for imaging through turbid layer with a large field of view. In our setup, a fluorescence sample is sandwiched between two turbid layers. Instead of generating one focus spot via wavefront shaping, we use an unshaped beam to illuminate the turbid layer and generate an unknown speckle pattern at the target plane over a wide field of view. By tilting the input wavefront, we raster scan the unknown speckle pattern via the memory effect and capture the corresponding low-resolution fluorescence images through the turbid layer. Different from the wavefront-shaping-based single-spot scanning, the proposed approach employs many spots (i.e., speckles) in parallel for extending the field of view. Based on all captured images, we jointly recover the fluorescence object, the unknown optical transfer function of the turbid layer, the translated step size, and the unknown speckle pattern. Without direct access to the object plane or knowledge of the turbid layer, we demonstrate a 13-fold resolution gain through the turbid layer using the reported strategy. We also demonstrate the use of this technique to improve the resolution of a low numerical aperture objective lens allowing to obtain both large field of view and high resolution at the same time. The reported method provides insight for developing new fluorescence imaging platforms and may find applications in deep-tissue imaging. PMID:29359102

Plant abiotic stress diagnostic by laser induced chlorophyll fluorescence spectral analysis of in vivo leaf tissue of biofuel species

NASA Astrophysics Data System (ADS)

Gouveia-Neto, Artur S.; Silva, Elias A., Jr.; Costa, Ernande B.; Bueno, Luciano A.; Silva, Luciana M. H.; Granja, Manuela M. C.; Medeiros, Maria J. L.; Câmara, Terezinha J. R.; Willadino, Lilia G.

2010-02-01

Laser induced fluorescence is exploited to evaluate the effect of abiotic stresses upon the evolution and characteristics of in vivo chlorophyll emission spectra of leaves tissues of brazilian biofuel plants species(Saccharum officinarum and Jatropha curcas). The chlorophyll fluorescence spectra of 20 min predarkened intact leaves were studied employing several excitation wavelengths in the UV-VIS spectral region. Red(Fr) and far-red (FFr) chlorophyll fluorescence emission signals around 685 nm and 735 nm, respectively, were analyzed as a function of the stress intensity and the time of illumination(Kautsky effect). The Chl fluorescence ratio Fr/FFr which is a valuable nondestructive indicator of the chlorophyll content of leaves was investigated during a period of time of 30 days. The dependence of the Chl fluorescence ratio Fr/FFr upon the intensity of the abiotic stress(salinity) was examined. The results indicated that the salinity plays a major hole in the chlorophyll concentration of leaves in both plants spieces, with a significant reduction in the chlorophyll content for NaCl concentrations in the 25 - 200 mM range. The laser induced chlorophyll fluorescence analysis allowed detection of damage caused by salinity in the early stages of the plants growing process, and can be used as an early-warning indicator of salinity stress

Layer-by-layer introduction of poly(phenylenevinylene) onto microspheres and probing the influence from the weak/strong polyanion spacer-layers.

PubMed

Song, Jing; Qiu, Tian; Chen, Yun; Zhang, Wei; Fan, Li-Juan

2015-08-15

The layer-by-layer (LBL) technique was employed for preparing fluorescent microspheres with a core-shell structure by the alternating adsorption of positively charged poly(p-phenylenevinylene) precursor (pre-PPV) and the polyanions onto polymer substrate spheres, followed by the thermal elimination to convert pre-PPV into fluorescent poly(p-phenylenevinylene) (PPV). Weak polyelectrolytes poly(acrylic acid) (PAA) (usually in a partly ionized form) and strong polyelectrolytes poly(sodium-p-styrenesulfonate) (PSS) were used as the anions to space the PPV layers and reduce the fluorescence self-quenching. Flow cytometry, combined with spectroscopy and microscopy, were used to study the structure and photophysical properties of the resulting microspheres. Optimization of the processing factors was carried out. PAA and PSS as weak and strong polyelectrolytes, respectively, displayed very different influence on the final emission of the spheres. Such difference was attributed to different inherent characteristics of PAA and PSS after detailed investigation in many aspects. In addition, the fluorescent spheres were found to have excellent photostability and thermal stability. Copyright © 2015 Elsevier Inc. All rights reserved.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Larsson, Annika; Soederberg, Linda; Westermark, Gunilla T.

Medin amyloid affects the medial layer of the thoracic aorta of most people above 50 years of age. The consequences of this amyloid are not completely known but the deposits may contribute to diseases such as thoracic aortic aneurysm and dissection or to the general diminished elasticity of blood vessels seen in elderly people. We show that the 50-amino acid residue peptide medin forms amyloid-like fibrils in vitro. With the use of Congo red staining, Thioflavin T fluorescence, electron microscopy, and a solid-phase binding assay on different synthetic peptides, we identified the last 18-19 amino acid residues to constitute themore » amyloid-promoting region of medin. We also demonstrate that the two C-terminal phenylalanines, previously suggested to be of importance for amyloid formation, are not required for medin amyloid formation.« less

A single dual-emissive nanofluorophore test paper for highly sensitive colorimetry-based quantification of blood glucose.

PubMed

Huang, Xiaoyan; Zhou, Yujie; Liu, Cui; Zhang, Ruilong; Zhang, Liying; Du, Shuhu; Liu, Bianhua; Han, Ming-Yong; Zhang, Zhongping

2016-12-15

Fluorescent test papers are promising for the wide applications in the assays of diagnosis, environments and foods, but unlike classical dye-absorption-based pH test paper, they are usually limited in the qualitative yes/no type of detection by fluorescent brightness, and the colorimetry-based quantification remains a challenging task. Here, we report a single dual-emissive nanofluorophore probe to achieve the consecutive color variations from blue to red for the quantification of blood glucose on its as-prepared test papers. Red quantum dots were embedded into silica nanoparticles as a stable internal standard emission, and blue carbon dots (CDs) were further covalently linked onto the surface of silica, in which the ratiometric fluorescence intensity of blue to red is controlled at 5:1. While the oxidation of glucose induced the formation of Fe(3+) ions, the blue emission of CDs was thus quenched by the electron transfer from CDs to Fe(3+), displaying a serial of consecutive color variations from blue to red with the dosage of glucose. The high-quality test papers printed by the probe ink exhibited a dosage-sensitive allochromatic capability with the clear differentiations of ~5, 7, 9, 11mM glucose in human serum (normal: 3-8mM). The blood glucose determined by the test paper was almost in accordance with that measured by a standard glucometer. The method reported here opens a window to the wide applications of fluorescent test paper in biological assays. Copyright © 2016 Elsevier B.V. All rights reserved.

Detection of irradiation induced reactive oxygen species production in live cells

NASA Astrophysics Data System (ADS)

Gao, Bo; Zhu, Debin

2006-09-01

Reactive oxygen species (ROS) is thought to play an important role in cell signaling of apoptosis, necrosis, and proliferation. Light irradiation increases mitochondrial reactive oxygen species (ROS) production and mediates its intracellular signaling by adjusting the redox potential in tumor cells. Mitochondria are the main source of ROS in the living cell. Superoxide anions (0 II - are likely the first ROS generated in the mitochondria following radiation damage, and then convert to hydrogen peroxide (H II0 II), hydroxyl radical (•OH), and singlet oxygen (10 II), etc. Conventional methods for research ROS production in mitochondria mostly use isolated mitochondria rather than mitochondria in living cells. In this study, a highly selective probe to detect mitochondrial 0 II - in live cells, MitoSOX TM Red, was applied to quantify the mitochondrial ROS production in human lung adenocarcinoma cells (ASTC-a-1) with laser scanning microscope (LSM) after ultraviolet C (UVC) and He-Ne laser irradiation. Dichiorodihydrofluoresein diacetate (DCFHDA), a common used fluorescent probe for ROS detection without specificity, were used as a comparison to image the ROS production. The fluorescent image of MItoSOX TM Red counterstained with MitoTracker Deep Red 633, a mitochondria selective probe, shows that the mitochondrial ROS production increases distinctly after UVC and He-Ne laser irradiation. DCFH-DA diffuses labeling throughout the cell though its fluorescence increases markedly too. In conclusion, the fluorescent method with MitoSOX TM Red reagent is proved to be a promising technique to research the role of ROS in radiation induced apoptosis.

BSA Au clusters as a probe for enhanced fluorescence detection using multipulse excitation scheme.

PubMed

Raut, Sangram L; Rich, Ryan; Fudala, Rafal; Kokate, R; Kimball, J D; Borejdo, Julian; Vishwanatha, Jamboor K; Gryczynski, Zygmunt; Gryczynski, Ignacy

2014-01-01

Although BSA Au clusters fluoresce in red region (λmax: 650 nm), they are of limited use due to low fluorescence quantum yield (~6%). Here we report an enhanced fluorescence imaging application of fluorescent bio-nano probe BSA Au clusters using multipulse excitation scheme. Multipulse excitation takes advantage of long fluorescence lifetime (> 1 µs) of BSA Au clusters and enhances its fluorescence intensity 15 times over short lived cellular auto-fluorescence. Moreover we have also shown that by using time gated detection strategy signal (fluorescence of BSA Au clusters) to noise (auto-fluorescence) ratio can be increased by 30 fold. Thereby with multipulse excitation long lifetime probes can be used to develop biochemical assays and perform optical imaging with zero background.

Photomultiplier window materials under electron irradiation - Fluorescence and phosphorescence

NASA Technical Reports Server (NTRS)

Viehmann, W.; Eubanks, A. G.; Pieper, G. F.; Bredekamp, J. H.

1975-01-01

The fluorescence and phosphorescence of photomultiplier window materials under electron irradiation have been investigated using a Sr-90/Y-90 beta emitter as the electron source. Spectral emission curves of UV-grade, optical-grade, and electron-irradiated samples of MgF2 and LiF, and of CaF2, BaF2, sapphire, fused silica, and UV-transmitting glasses were obtained over the 200-650-nm spectral range. Fluorescence yields were determined on these materials utilizing photomultiplier tubes with cesium telluride, bialkali, and trialkali (S-20) photocathodes, respectively. Optical-grade MgF2 and LiF, as well as electron-irradiated UV-grade samples of these two materials, show enhanced fluorescence due to color-center formation and associated emission bands in the blue and red wavelength regions. Large variations in fluorescence intensities were found in UV-grade sapphire samples of different origins, particularly in the red end of the spectrum, presumably due to various amounts of chromium-ion content. Phosphorescence decay with time is best described by a sum of exponential terms, with time constants ranging from a few minutes to several days.

Novel type of red-shifted chlorophyll a antenna complex from Chromera velia. I. Physiological relevance and functional connection to photosystems.

PubMed

Kotabová, Eva; Jarešová, Jana; Kaňa, Radek; Sobotka, Roman; Bína, David; Prášil, Ondřej

2014-06-01

Chromera velia is an alveolate alga associated with scleractinian corals. Here we present detailed work on chromatic adaptation in C. velia cultured under either blue or red light. Growth of C. velia under red light induced the accumulation of a light harvesting antenna complex exhibiting unusual spectroscopic properties with red-shifted absorption and atypical 710nm fluorescence emission at room temperature. Due to these characteristic features the complex was designated "Red-shifted Chromera light harvesting complex" (Red-CLH complex). Its detailed biochemical survey is described in the accompanying paper (Bina et al. 2013, this issue). Here, we show that the accumulation of Red-CLH complex under red light represents a slow acclimation process (days) that is reversible with much faster kinetics (hours) under blue light. This chromatic adaptation allows C. velia to maintain all important parameters of photosynthesis constant under both light colors. We further demonstrated that the C. velia Red-CLH complex is assembled from a 17kDa antenna protein and is functionally connected to photosystem II as it shows variability of chlorophyll fluorescence. Red-CLH also serves as an additional locus for non-photochemical quenching. Although overall rates of oxygen evolution and carbon fixation were similar for both blue and red light conditions, the presence of Red-CLH in C. velia cells increases the light harvesting potential of photosystem II, which manifested as a doubled oxygen evolution rate at illumination above 695nm. This data demonstrates a remarkable long-term remodeling of C. velia light-harvesting system according to light quality and suggests physiological significance of 'red' antenna complexes. Copyright © 2014 Elsevier B.V. All rights reserved.

Persistent luminescence nanothermometers

NASA Astrophysics Data System (ADS)

Martín Rodríguez, Emma; López-Peña, Gabriel; Montes, Eduardo; Lifante, Ginés; García Solé, José; Jaque, Daniel; Diaz-Torres, Luis Armando; Salas, Pedro

2017-08-01

Persistent phosphorescence nanoparticles emitting in the red and near-infrared spectral regions are strongly demanded as contrast nanoprobes for autofluorescence free bioimaging and biosensing. In this work, we have developed Sr4Al14O25:Eu2+, Cr3+, Nd3+ nanopowders that produce persistent red phosphorescence peaking at 694 nm generated by Cr3+ ions. This emission displays temperature sensitivity in the physiological temperature range (20-60 °C), which makes these nanoparticles potentially useful as fluorescence (contactless) nanothermometers operating without requiring optical excitation. Nd3+ ions, which act as shallow electron traps for the red Cr3+ persistent emission, also display infrared emission bands, extending the fluorescence imaging capability to the second biological window. This unique combination of properties makes these nanoparticles multifunctional luminescent probes with great potential applications in nanomedicine.

Comparative assessment of fluorescent proteins for in vivo imaging in an animal model system.

PubMed

Heppert, Jennifer K; Dickinson, Daniel J; Pani, Ariel M; Higgins, Christopher D; Steward, Annette; Ahringer, Julie; Kuhn, Jeffrey R; Goldstein, Bob

2016-11-07

Fluorescent protein tags are fundamental tools used to visualize gene products and analyze their dynamics in vivo. Recent advances in genome editing have expedited the precise insertion of fluorescent protein tags into the genomes of diverse organisms. These advances expand the potential of in vivo imaging experiments and facilitate experimentation with new, bright, photostable fluorescent proteins. Most quantitative comparisons of the brightness and photostability of different fluorescent proteins have been made in vitro, removed from biological variables that govern their performance in cells or organisms. To address the gap, we quantitatively assessed fluorescent protein properties in vivo in an animal model system. We generated transgenic Caenorhabditis elegans strains expressing green, yellow, or red fluorescent proteins in embryos and imaged embryos expressing different fluorescent proteins under the same conditions for direct comparison. We found that mNeonGreen was not as bright in vivo as predicted based on in vitro data but is a better tag than GFP for specific kinds of experiments, and we report on optimal red fluorescent proteins. These results identify ideal fluorescent proteins for imaging in vivo in C. elegans embryos and suggest good candidate fluorescent proteins to test in other animal model systems for in vivo imaging experiments. © 2016 Heppert et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

Effects of alcohols on fluorescence intensity and color of a discharged-obelin-based biomarker.

PubMed

Alieva, Roza R; Belogurova, Nadezhda V; Petrova, Alena S; Kudryasheva, Nadezhda S

2014-05-01

Photoproteins are responsible for bioluminescence of marine coelenterates; bioluminescent and fluorescent biomarkers based on photoproteins are useful for monitoring of calcium-dependent processes in medical investigations. Here, we present the analysis of intensity and color of light-induced fluorescence of Ca(2+)-discharged photoprotein obelin in the presence of alcohols (ethanol and glycerol). Complex obelin spectra obtained at different concentrations of the alcohols at 350- and 280-nm excitation (corresponding to polypeptide-bound coelenteramide and tryptophan absorption regions) were deconvoluted into Gaussian components; fluorescent intensity and contributions of the components to experimental spectra were analyzed. Five Gaussian components were found in different spectral regions-ultraviolet (tryptophan emission), blue-green (coelenteramide emission), and red (hypothetical indole-coelenteramide exciplex emission). Inhibition coefficients and contributions of the components to experimental fluorescent spectra showed that presence of alcohols increased contributions of ultraviolet, violet, and red components, but decreased contributions of components in the blue-green region. The effects were related to (1) changes of proton transfer efficiency in fluorescent S*1 state of coelenteramide in the obelin active center and (2) formation of indole-coelenteramide exciplex at 280-nm photoexcitation. The data show that variation of fluorescence color and intensity in the presence of alcohols and dependence of emission spectra on excitation wavelength should be considered while applying the discharged obelin as a fluorescence biomarker.

Incubation and application of transgenic green fluorescent nude mice in visualization studies on glioma tissue remodeling.

PubMed

Dong, Jun; Dai, Xing-liang; Lu, Zhao-hui; Fei, Xi-feng; Chen, Hua; Zhang, Quan-bin; Zhao, Yao-dong; Wang, Zhi-min; Wang, Ai-dong; Lan, Qing; Huang, Qiang

2012-12-01

The primary reasons for local recurrence and therapeutic failure in the treatment of malignant gliomas are the invasion and interactions of tumor cells with surrounding normal brain cells. However, these tumor cells are hard to be visualized directly in histopathological preparations, or in experimental glioma models. Therefore, we developed an experimental human dual-color in vivo glioma model, which made tracking solitary invasive glioma cells possible, for the purpose of visualizing the interactions between red fluorescence labeled human glioma cells and host brain cells. This may offer references for further studying the roles of tumor microenvironment during glioma tissue remodeling. Transgenic female C57BL/6 mice expressing enhanced green fluorescent protein (EGFP) were crossed with male Balb/c nude mice. Then sib mating was allowed to occur continuously in order to establish an inbred nude mice strain with 50% of their offspring that are EGFP positive. Human glioma cell lines U87-MG and SU3 were transfected with red fluorescent protein (RFP) gene, and a rat C6 glioma cell line was stained directly with CM-DiI, to establish three glioma cell lines emitting red fluorescence (SU3-RFP, U87-RFP, and C6-CM-DiI). Red fluorescence tumor cells were inoculated via intra-cerebral injection into caudate nucleus of the EGFP nude mice. Tumor-bearing mice were sacrificed when their clinical symptoms appeared, and the whole brain was harvested and snap frozen for further analysis. Confocal laser scanning microscopy was performed to monitor the mutual interactions between tumor cells and host brain cells. Almost all the essential tissues of the established EGFP athymic Balb/c nude mice, except hair and erythrocytes, fluoresced green under excitation using a blue light-emitting flashlight with a central peak of 470 nm, approximately 50% of the offsprings were nu/nu EGFP+. SU3-RFP, U87-RFP, and C6-CM-DiI almost 100% expressed red fluorescence under the fluorescence microscope. Under fluorescence microscopic view, RFP+ cells were observed growing wherever they arrived at, locating in the brain parenchyma, ventricles, and para-vascular region. The interactions between the transplanted tumor cells and host adjacent cells could be classified into three types: (1) interweaving; (2) mergence; and (3) fusion. Interweaving was observed in the early stage of tumor remodeling, in which both transplantable tumor cells and host cells were observed scattered in the tumor invading and spreading area without organic connections. Mergence was defined as mutual interactions between tumor cells and host stroma during tumorigenesis. Direct cell fusion between transplantable tumor cells and host cells could be observed occasionally. This study showed that self-established EGFP athymic nude mice offered the possibility of visualizing tumorigenesis of human xenograft tumor, and the dual-color xenograft glioma model was of considerable utility in studying the process of tumor remodeling. Based on this platform, mutual interactions between glioma cells and host tissues could be observed directly to further elucidate the development of tumor microenvironment.

Application of the shifted excitation Raman difference spectroscopy (SERDS) to the analysis of trace amounts of methanol in red wines

NASA Astrophysics Data System (ADS)

Volodin, Boris; Dolgy, Sergei; Ban, Vladimir S.; Gracin, Davor; Juraić, Krunoslav; Gracin, Leo

2014-03-01

Shifted Excitation Raman Difference Spectroscopy (SERDS) has proven an effective method for performing Raman analysis of fluorescent samples. This technique allows achieving excellent signal to noise performance with shorter excitation wavelengths, thus taking full advantage of the superior signal strength afforded by shorter excitation wavelengths and the superior performance, also combined with lower cost, delivered by silicon CCDs. The technique is enabled by use of two closely space fixed-wavelength laser diode sources stabilized with the Volume Bragg gratings (VBGs). A side by side comparison reveals that SERDS technique delivers superior signal to noise ratio and better detection limits in most situations, even when a longer excitation wavelength is employed for the purpose of elimination of the fluorescence. We have applied the SERDS technique to the quantitative analysis of the presence of trace amounts of methanol in red wines, which is an important task in quality control operations within wine industry and is currently difficult to perform in the field. So far conventional Raman spectroscopy analysis of red wines has been impractical due to the high degree of fluorescence.

In-vivo fluorescence detection and imaging of porphyrin-producing bacteria in the human skin and in the oral cavity for diagnosis of acne vulgaris, caries, and squamous cell carcinoma

NASA Astrophysics Data System (ADS)

Koenig, Karsten; Schneckenburger, Herbert; Hemmer, Joerg; Tromberg, Bruce J.; Steiner, Rudolf W.

1994-05-01

Certain bacteria are able to synthesize metal-free fluorescent porphyrins and can therefore be detected by sensitive autofluorescence measurements in the red spectral region. The porphyrin-producing bacterium Propionibacterium acnes, which is involved in the pathogenesis of acne vulgaris, was localized in human skin. Spectrally resolved fluorescence images of bacteria distribution in the face were obtained by a slow-scan CCD camera combined with a tunable liquid crystal filter. The structured autofluorescence of dental caries and dental plaque in the red is caused by oral bacteria, like Bacteroides or Actinomyces odontolyticus. `Caries images' were created by time-gated imaging in the ns-region after ultrashort laser excitation. Time-gated measurements allow the suppression of backscattered light and non-porphyrin autofluorescence. Biopsies of oral squamous cell carcinoma exhibited red autofluorescence in necrotic regions and high concentrations of the porphyrin-producing bacterium Pseudomonas aerigunosa. These studies suggest that the temporal and spectral characteristics of bacterial autofluorescence can be used in the diagnosis and treatment of a variety of diseases.

Highly efficient organic light emitting diodes formed by solution processed red emitters with evaporated blue common layer structure.

PubMed

Cho, Ye Ram; Kim, Hyung Suk; Yu, Young-Jun; Suh, Min Chul

2015-10-30

We prepared highly-efficient solution-processed red phosphorescent organic light emitting diodes (PHOLEDs) with a blue common layer structure that can reasonably confine the triplet excitons inside of the red emission layer (EML) with the assistance of a bipolar exciton blocking layer. The red PHOLEDs containing EML with a 7 : 3 ratio of 11-(4,6-diphenyl-[1,3,5]triazin-2-yl)-12-phenyl-11,12-dihydro-11,12-diaza-indeno[2,1-a]fluorene (n-type host, NH) : 4-(3-(triphenylen-2-yl)phenyl)dibenzo[b,d]thiophene (p-type host, PH) doped with 5% Iridium(III) bis(2-(3,5-dimethylphenyl)quinolinato-N,C2')tetramethylheptadionate (Red Dopant, RD) produced the highest current and power efficiencies at 23.4 cd/A and 13.6 lm/W, with a 19% external quantum efficiency at 1000 cd/m(2). To the best of our knowledge, such efficiency was the best among those that have been obtained from solution-processed small molecular red PHOLEDs. In addition, the host molecules utilized in this study have no flexible spacers, such as an alkyl chain, which normally deteriorate the stability of the device.

Highly efficient organic light emitting diodes formed by solution processed red emitters with evaporated blue common layer structure

NASA Astrophysics Data System (ADS)

Cho, Ye Ram; Kim, Hyung Suk; Yu, Young-Jun; Suh, Min Chul

2015-10-01

We prepared highly-efficient solution-processed red phosphorescent organic light emitting diodes (PHOLEDs) with a blue common layer structure that can reasonably confine the triplet excitons inside of the red emission layer (EML) with the assistance of a bipolar exciton blocking layer. The red PHOLEDs containing EML with a 7 : 3 ratio of 11-(4,6-diphenyl-[1,3,5]triazin-2-yl)-12-phenyl-11,12-dihydro-11,12-diaza-indeno[2,1-a]fluorene (n-type host, NH) : 4-(3-(triphenylen-2-yl)phenyl)dibenzo[b,d]thiophene (p-type host, PH) doped with 5% Iridium(III) bis(2-(3,5-dimethylphenyl)quinolinato-N,C2’)tetramethylheptadionate (Red Dopant, RD) produced the highest current and power efficiencies at 23.4 cd/A and 13.6 lm/W, with a 19% external quantum efficiency at 1000 cd/m2. To the best of our knowledge, such efficiency was the best among those that have been obtained from solution-processed small molecular red PHOLEDs. In addition, the host molecules utilized in this study have no flexible spacers, such as an alkyl chain, which normally deteriorate the stability of the device.

Highly efficient organic light emitting diodes formed by solution processed red emitters with evaporated blue common layer structure

PubMed Central

Cho, Ye Ram; Kim, Hyung Suk; Yu, Young-Jun; Suh, Min Chul

2015-01-01

We prepared highly-efficient solution-processed red phosphorescent organic light emitting diodes (PHOLEDs) with a blue common layer structure that can reasonably confine the triplet excitons inside of the red emission layer (EML) with the assistance of a bipolar exciton blocking layer. The red PHOLEDs containing EML with a 7 : 3 ratio of 11-(4,6-diphenyl-[1,3,5]triazin-2-yl)-12-phenyl-11,12-dihydro-11,12-diaza-indeno[2,1-a]fluorene (n-type host, NH) : 4-(3-(triphenylen-2-yl)phenyl)dibenzo[b,d]thiophene (p-type host, PH) doped with 5% Iridium(III) bis(2-(3,5-dimethylphenyl)quinolinato-N,C2’)tetramethylheptadionate (Red Dopant, RD) produced the highest current and power efficiencies at 23.4 cd/A and 13.6 lm/W, with a 19% external quantum efficiency at 1000 cd/m2. To the best of our knowledge, such efficiency was the best among those that have been obtained from solution-processed small molecular red PHOLEDs. In addition, the host molecules utilized in this study have no flexible spacers, such as an alkyl chain, which normally deteriorate the stability of the device. PMID:26514274

Characterization of marine macroalgae by fluorescence signatures

NASA Technical Reports Server (NTRS)

Topinka, J. A.; Bellows, W. Korjeff; Yentsch, C. S.

1990-01-01

The feasibility of distinguishing macroalgal classes by their fluorescence signatures was investigated using narrow-waveband light to excite groups of accessory pigments in brown, red, and green macroalgae and measuring fluorescence emission at 685 nm. Results obtained on 20 marine macroalgae field-collected samples showed that fluorescence excitation signatures were relatively uniform within phylogenetic classes but were substantially different for different classes. It is suggested that it may be possible to characterize the type and the abundance of subtidal macroalgae from low-flying aircraft using existing laser-induced fluorescence methodology.

Nanostructured fluorescent particles for glucose sensing

NASA Astrophysics Data System (ADS)

Grant, Patrick S.; Fang, Ming; Lvov, Yuri; McShane, Michael J.

2002-05-01

Self-assembled thin films containing embedded enzymes and fluorescent indicators are being developed for use as highly specific glucose biosensors. The sensors are fabricated using electrostatic Layer-by-Layer (LBL) adsorption to create oxygen-sensitive (Ruthenium-based) layers, the fluorescent intensity of which responds to changes in local oxygen levels. Oxygen is consumed locally by the reaction between glucose oxidase (GOx) molecules and glucose. Latex particles serve as the templates for our sensors and fabrication is carried out through the alternate adsorption of multiple levels of {GOx/polycation} and {Ruthenium-polycation/polyanion} bilayers. Additional fluorescence layers as well as fluorescent latex are being considered as internal intensity references to allow ratiometric monitoring. Films adsorbed to the nanoparticle templates are being studied to understand the fundamental chemical and optical properties, including enzymatic activity, spectral shape and emission intensity. Enzymatic activity is retained and stability is improved after adsorption, and increased surface area afforded by the particles allows use of increased numbers of molecules. Fluorescence is also maintained, though blue shifts are observed in emission spectra, and indicator activity remains. In vitro characterization studies demonstrate the feasibility of the particles as glucose biosensors, and future work will aim to optimize the response for neural monitoring.

Physical Mechanisms Routing Nutrients in the Central Red Sea

NASA Astrophysics Data System (ADS)

Zarokanellos, Nikolaos D.; Kürten, Benjamin; Churchill, James H.; Roder, Cornelia; Voolstra, Christian R.; Abualnaja, Yasser; Jones, Burton H.

2017-11-01

Mesoscale eddies and boundary currents play a key role in the upper layer circulation of the Red Sea. This study assesses the physical and biochemical characteristics of an eastern boundary current (EBC) and recurrent eddies in the central Red Sea (CRS) using a combination of in situ and satellite observations. Hydrographic surveys in November 2013 (autumn) and in April 2014 (spring) in the CRS (22.15°N-24.1°N) included a total of 39 and 27 CTD stations, respectively. In addition, high-resolution hydrographic data were acquired in spring 2014 with a towed undulating vehicle (ScanFish). In situ measurements of salinity, temperature, chlorophyll fluorescence, colored dissolved organic matter (CDOM), and dissolved nitrate: phosphorous ratios reveal distinct water mass characteristics for the two periods. An EBC, observed in the upper 150 m of the water column during autumn, transported low-salinity and warm water from the south toward the CRS. Patches of the low-salinity water of southern origin tended to contain relatively high concentrations of chlorophyll and CDOM. The prominent dynamic feature observed in spring was a cyclonic/anticyclonic eddy pair. The cyclonic eddy was responsible for an upward nutrient flux into the euphotic zone. Higher chlorophyll and CDOM concentrations, and concomitant lower nitrate:phosphorous ratios indicate the influence of the EBC in the CRS at the end of the stratified summer period.

A passive two-band sensor of sunlight-excited plant fluorescence

NASA Astrophysics Data System (ADS)

Kebabian, Paul L.; Theisen, Arnold F.; Kallelis, Spiros; Freedman, Andrew

1999-11-01

We have designed and built a passive remote sensor of sunlight-excited chlorophyll fluorescence (U.S. Patent No. 5,567,947, Oct. 22, 1996) which provides for the real-time, in situ sensing of photosynthetic activity in plants. This sensor, which operates as a Fraunhofer line discriminator, detects light at the cores of the lines comprising the atmospheric oxygen A and B bands, centered at 762 and 688 nm, respectively. These bands also correspond to wavelengths in the far-red and red chlorophyll fluorescence bands. The sensor is based on an induced fluorescence approach; as light collected from fluorescing plants is passed through a low-pressure cell containing oxygen, the oxygen absorbs the energy and subsequently reemits photons which are then detected by a photomultiplier tube. Since the oxygen in the cell absorbs light at the same wavelengths that have been strongly absorbed by the oxygen in the atmosphere, the response to incident sunlight is minimal. This mode of measurement is limited to target plants sufficiently close in range that the plants' fluorescence is not itself appreciably absorbed by atmospheric oxygen (˜200 m). In vivo measurements of fluorescence in the 760 and 690 nm bands of vegetation in full sunlight are also presented. Measurements of plant fluorescence at the single-plant canopy level were obtained from greenhouse-grown bean plants subjected to a range of nitrogen treatments. The ratio of the fluorescence obtained from the two measurement bands showed statistically significant variation with respect to nitrogen treatments.

Intraoperative Fluorescence Cerebral Angiography by Laser Surgical Microscopy: Comparison With Xenon Microscopy and Simultaneous Observation of Cerebral Blood Flow and Surrounding Structures.

PubMed

Ito, Yuhei; Suzuki, Kyouichi; Ichikawa, Tsuyoshi; Watanabe, Yoichi; Sato, Taku; Sakuma, Jun; Saito, Kiyoshi

2018-06-12

Laser surgical microscopes should enable uniform illumination of the operative field, and require less luminous energy compared with existing xenon surgical microscopes. To examine the utility of laser illumination in fluorescence cerebral angiography. Fluorescein sodium (fluorescein) was used as a fluorescent dye. We first compared the clarity of cerebral blood flow images collected by fluorescence angiography between the laser illumination and xenon illumination methods. We then assessed use of the laser illuminator for simultaneous observation of blood flow and surrounding structures during fluorescence angiography. Furthermore, the study was designed to evaluate usefulness of the thus determined excitation light in clinical cases. Fluorescence angiography using blue light laser for excitation provided higher clarity and contrast blood flow images compared with using blue light generated from a xenon lamp. Further, illumination with excitation light consisting of a combination of 3 types of laser (higher level of blue light, no green light, and lower level of red light) enabled both blood flow and surrounding structures to be observed through the microscope directly by the surgeon. Laser-illuminated fluorescence angiography provides high clarity and contrast images of cerebral blood flow. Further, a laser providing strong blue light and weak red light for excitation light enables simultaneous visual observation of fluorescent blood flow and surrounding structures by the surgeon using a surgical microscope. Overall, these data suggest that laser surgical microscopes are useful for both ordinary operative manipulations and fluorescence angiography.

Biomimetic Mineralization of Gold Nanoclusters as Multifunctional Thin Films for Glass Nanopore Modification, Characterization, and Sensing.

PubMed

Cao, Sumei; Ding, Shushu; Liu, Yingzi; Zhu, Anwei; Shi, Guoyue

2017-08-01

Hurdles of nanopore modification and characterization restrain the development of glass capillary-based nanopore sensing platforms. In this article, a simple but effective biomimetic mineralization method was developed to decorate glass nanopore with a thin film of bovine serum albumin-protected Au nanocluster (BSA-Au NC). The BSA-Au NC film emitted a strong red fluorescence whereby nondestructive characterization of Au film decorated at the inner surface of glass nanopore can be facilely achieved by a fluorescence microscopy. Besides, the BSA molecules played dual roles in the fabrication of functionalized Au thin film in glass nanopore: they not only directed the synthesis of fluorescent Au thin film but also provided binding sites for recognition, thus achieving synthesis-modification integration. This occurred due to the ionized carboxyl groups (-COO - ) of a BSA coating layer on Au NCs which can interacted with arginine (Arg) via guanidinium groups. The added Arg selectively led to the change in the charge and ionic current of BSA-Au NC film-decorated glass nanopore. Such ionic current responses can be used for quantifying Arg with a detection limit down to 1 fM, which was more sensitive than that of previous sensing systems. Together, the designed method exhibited great promise in providing a facile and controllable solution for glass nanopore modification, characterization, and sensing.

Stoke's and anti-Stoke's characteristics of anaerobic and aerobic bacterias at excitation of fluorescence by low-intensity red light: I. Research of anaerobic bacterias

NASA Astrophysics Data System (ADS)

Masychev, Victor I.; Alexandrov, Michail T.

2000-04-01

Biopsy or photo dynamic therapy of tumors are usually investigated by fluorescent diagnostics methods. Information on modified method of fluorescence diagnostics of inflammatory diseases is represented in this research. Anaerobic micro organisms are often the cause of these pathological processes. These micro organisms also accompany disbiotic processes in intestines.

Acrodynia: exposure to mercury from fluorescent light bulbs

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tunnessen, W.W. Jr.; McMahon, K.J.; Baser, M.

1987-05-01

Medical attention was sought for a 23-month-old toddler because of anorexia, weight loss, irritability, profuse sweating, peeling and redness of his fingers and toes, and a miliarial rash. The diagnosis was mercury poisoning, and an investigation of his environment disclosed that he had been exposed to mercury from broken fluorescent light bulbs. Acrodynia resulting from fluorescent bulbs has not been previously reported.

High pressure liquid chromatographic determination of aflatoxins in spices.

PubMed

Awe, M J; Schranz, J L

1981-11-01

High pressure liquid chromatography with fluorescence detection is used to determine aflatoxin in 5 common spices. A 10 micrometer microparticulate silica gel column is used with a dichloromethane-cyclohexane-acetonitrile solvent system to resolve aflatoxins B1, G1, B2, and G2. The fluorescence detector contained a silica gel-packed flowcell. Samples of black, white, and red pepper, ginger, and nutmeg were extracted according to a previously published method. Recoveries from aflatoxin-free samples of white pepper, ginger, and red pepper spiked with 1-50 micrograms aflatoxin/kg ranged from 64 to 92%.

Stacked white OLED having separate red, green and blue sub-elements

DOEpatents

Forrest, Stephen; Qi, Xiangfei; Slootsky, Michael

2014-07-01

The present invention relates to efficient organic light emitting devices (OLEDs). The devices employ three emissive sub-elements, typically emitting red, green and blue, to sufficiently cover the visible spectrum. Thus, the devices may be white-emitting OLEDs, or WOLEDs. Each sub-element comprises at least one organic layer which is an emissive layer--i.e., the layer is capable of emitting light when a voltage is applied across the stacked device. The sub-elements are vertically stacked and are separated by charge generating layers. The charge-generating layers are layers that inject charge carriers into the adjacent layer(s) but do not have a direct external connection.

Direct liquid chromatography method for the simultaneous quantification of hydroxytyrosol and tyrosol in red wines.

PubMed

Piñeiro, Zulema; Cantos-Villar, Emma; Palma, Miguel; Puertas, Belen

2011-11-09

A validated HPLC method with fluorescence detection for the simultaneous quantification of hydroxytyrosol and tyrosol in red wines is described. Detection conditions for both compounds were optimized (excitation at 279 and 278 and emission at 631 and 598 nm for hydroxytyrosol and tyrosol, respectively). The validation of the analytical method was based on selectivity, linearity, robustness, detection and quantification limits, repeatability, and recovery. The detection and quantification limits in red wines were set at 0.023 and 0.076 mg L(-1) for hydroxytyrosol and at 0.007 and 0.024 mg L(-1) for tyrosol determination, respectively. Precision values, both within-day and between-day (n = 5), remained below 3% for both compounds. In addition, a fractional factorial experimental design was developed to analyze the influence of six different conditions on analysis. The final optimized HPLC-fluorescence method allowed the analysis of 30 nonpretreated Spanish red wines to evaluate their hydroxytyrosol and tyrosol contents.

Highly Efficient Red-Emitting Carbon Dots with Gram-Scale Yield for Bioimaging.

PubMed

Ding, Hui; Wei, Ji-Shi; Zhong, Ning; Gao, Qing-Yu; Xiong, Huan-Ming

2017-11-07

Carbon dots (CDs) are a new class of photoluminescent (PL), biocompatible, environment-friendly, and low-cost carbon nanomaterials. Synthesis of highly efficient red-emitting carbon dots (R-CDs) on a gram scale is a great challenge at present, which heavily restricts the wide applications of CDs in the bioimaging field. Herein, R-CDs with a high quantum yield (QY) of 53% are produced on a gram scale by heating a formamide solution of citric acid and ethylenediamine. The as-prepared R-CDs have an average size of 4.1 nm and a nitrogen content of about 30%, with an excitation-independent emission at 627 nm. After detailed characterizations, such strong red fluorescence is ascribed to the contribution from the nitrogen- and oxygen-related surface states and the nitrogen-derived structures in the R-CD cores. Our R-CDs show good photostability and low cytotoxicity, and thus they are excellent red fluorescence probes for bioimaging both in vitro and in vivo.

Fluorescent carbon dots: facile synthesis at room temperature and its application for Fe2+ sensing

NASA Astrophysics Data System (ADS)

Xiao, Sai Jin; Chu, Zhao Jun; Zuo, Jun; Zhao, Xiao Jing; Huang, Cheng Zhi; Zhang, Li

2017-02-01

A new route for one-pot preparation of carbon dots (CDs) was developed at room temperature using PEG400 as both the carbon source and passitive agent. The new method possesses the advantages of facile, rapid, energy-saving, without any external stimulus and environment friendly. By changing the content of NaOH, the PEG400-CDs with blue-emitting, yellow-emitting, orange red-emitting and red-emitting were obtained, and the formation mechanism were carefully investigated. In addition, a sensitive fluorescence sensor were developed for Fe2+ detection based on PEG400-CDs since the fluorescence of PEG400-CDs could be enhanced by Fe2+. It was found that there is a good linear relationship between the enhanced fluorescence and Fe2+ concentration in the range of 0.5 to 2.0 μmol·L-1 with the detection limit of 6.0 × 10-8 mol·L-1, and Fe2+ in water samples was also determined with high accuracy and repeatability.

Deep-Red Fluorescent Gold Nanoclusters for Nucleoli Staining: Real-Time Monitoring of the Nucleolar Dynamics in Reverse Transformation of Malignant Cells.

PubMed

Wang, Xiaojuan; Wang, Yanan; He, Hua; Ma, Xiqi; Chen, Qi; Zhang, Shuai; Ge, Baosheng; Wang, Shengjie; Nau, Werner M; Huang, Fang

2017-05-31

Nucleoli are important subnuclear structures inside cells. We report novel fluorescent gold nanoclusters (K-AuNCs) that are able to stain the nucleoli selectively and make it possible to explore the nucleolar morphology with fluorescence imaging technique. This novel probe is prepared through an easy synthesis method by employing a tripeptide (Lys-Cys-Lys) as the surface ligand. The properties, including deep-red fluorescence emission (680 nm), large Stocks shift, broad excitation band, low cytotoxicity, and good photostability, endow this probe with potential for bioanalytical applications. Because of their small size and their positively charged surface, K-AuNCs are able to accumulate efficiently at the nucleolar regions and provide precise morphological information. K-AuNCs are also used to monitor the nucleolar dynamics along the reverse-transformation process of malignant cells, induced by the agonist of protein A, 8-chloro-cyclic adenosine monophosphate. This gives a novel approach for investigating the working mechanism of antitumor drugs.

Fluorescent lighting with aluminum nitride phosphors

DOEpatents

Cherepy, Nerine J.; Payne, Stephen A.; Seeley, Zachary M.; Srivastava, Alok M.

2016-05-10

A fluorescent lamp includes a glass envelope; at least two electrodes connected to the glass envelope; mercury vapor and an inert gas within the glass envelope; and a phosphor within the glass envelope, wherein the phosphor blend includes aluminum nitride. The phosphor may be a wurtzite (hexagonal) crystalline structure Al.sub.(1-x)M.sub.xN phosphor, where M may be drawn from beryllium, magnesium, calcium, strontium, barium, zinc, scandium, yttrium, lanthanum, cerium, praseodymium, europium, gadolinium, terbium, ytterbium, bismuth, manganese, silicon, germanium, tin, boron, or gallium is synthesized to include dopants to control its luminescence under ultraviolet excitation. The disclosed Al.sub.(1-x)M.sub.xN:Mn phosphor provides bright orange-red emission, comparable in efficiency and spectrum to that of the standard orange-red phosphor used in fluorescent lighting, Y.sub.2O.sub.3:Eu. Furthermore, it offers excellent lumen maintenance in a fluorescent lamp, and does not utilize "critical rare earths," minimizing sensitivity to fluctuating market prices for the rare earth elements.

Ecological changes in oral microcosm biofilm during maturation

NASA Astrophysics Data System (ADS)

Kim, Young-Seok; Kang, Si-Mook; Lee, Eun-Song; Lee, Ji Hyun; Kim, Bo-Ra; Kim, Baek-Il

2016-10-01

The aim of this study was to evaluate the ecological changes in the biofilm at different stages of maturation using 16S rDNA gene amplicon sequencing and to identify correlations between red/green (R/G) fluorescence ratio and ecological changes. An oral microcosm biofilm was initiated from the saliva of a single donor and grown anaerobically for up to 10 days in basal medium mucin. Quantitative light-induced fluorescence analysis was shown that the R/G ratio of the biofilm increased consistently, but the slope rapidly decreased after six days. The bacterial compositions of 10 species also consistently changed over time. However, there was no significant correlation between each bacteria and red fluorescence. The monitoring of the maturation process of oral microcosm biofilm over 10 days revealed that the R/G ratio and the bacterial composition within biofilm consistently changed. Therefore, the R/G fluorescence ratio of biofilm may be related with its ecological change rather than specific bacteria.

Tracking variations in fluorescent-dissolved organic matter in an aerobic submerged membrane bioreactor using excitation-emission matrix spectra combined with parallel factor analysis.

PubMed

Hur, Jin; Shin, Jaewon; Kang, Minsun; Cho, Jinwoo

2014-08-01

In this study, the variations in the fluorescent components of dissolved organic matter (DOM) were tracked for an aerobic submerged membrane bioreactor (MBR) at three different operation stages (cake layer formation, condensation, and after cleaning). The fluorescent DOM was characterized using excitation-emission matrix (EEM) spectroscopy combined with parallel factor analysis (PARAFAC). Non-aromatic carbon structures appear to be actively involved in the membrane fouling for the cake layer formation stage as revealed by much higher UV-absorbing DOM per organic carbon found in the effluent versus those inside the reactor. Four fluorescent components were successfully identified from the reactor and the effluent DOMs by EEM-PARAFAC modeling. Among those in the reactor, microbial humic-like fluorescence was the most abundant component at the cake layer formation stage and tryptophan-like fluorescence at the condensation stage. In contrast to the reactor, relatively similar composition of the PARAFAC components was exhibited for the effluent at all three stages. Tryptophan-like fluorescence displayed the largest difference between the reactor and the effluent, suggesting that this component could be a good tracer for membrane fouling. It appears that the fluorescent DOM was involved in membrane fouling by cake layer formation rather than by internal pore adsorption because its difference between the reactor and the effluent was the highest among all the four components, even after the membrane cleaning. Our study provided an insight into the fate and the behavior fluorescent DOM components for an MBR system, which could be an indicator of the membrane fouling.

Detection of altered extracellular matrix in surface layers of unstable carotid plaque: an optical spectroscopy, birefringence and microarray genetic analysis.

PubMed

Korol, Renee M; Canham, Peter B; Liu, Li; Viswanathan, Kasinath; Ferguson, Gary G; Hammond, Rob R; Finlay, Helen M; Baker, Henry V; Lopez, Cecilia; Lucas, Alexandra R

2011-01-01

Erosion and rupture of surface layers in atherosclerotic plaque can cause heart attack and stroke; however, changes in luminal surface composition are incompletely defined. Laser-induced fluorescence spectroscopy (LIFS), with limited tissue penetration, was used to investigate the surface of unstable carotid plaque and correlated with microscopy, birefringence and gene expression. Arterial matrix collagens I, III and elastin were assessed in unstable plaques (n = 25) and reference left internal mammary arteries (LIMA, n = 10). LIFS in addition to selective histological staining with picrosirius red, Movat pentachrome and immunostaining revealed decreased elastin and increased collagen I and III (P < 0.05) in carotid plaque when compared with LIMA. Within plaque, collagen I was elevated in the internal carotid region versus the common carotid region. Polarized light microscopy detected layers of aligned collagen and associated mechanical rigidity of the fibrous cap. Microarray analysis of three carotid and three LIMA specimens confirmed up-regulation of collagen I, III and IV, lysyl oxidase and MMP-12. In conclusion, LIFS analysis coupled with microscopy revealed marked regional differences in collagen I, III and elastin in surface layers of carotid plaque; indicative of plaque instability. Birefringence measurements demonstrated mechanical rigidity and weakening of the fibrous cap with complementary changes in ECM gene expression. © 2011 The Authors. Photochemistry and Photobiology © 2011 The American Society of Photobiology.

Interpretation of the fluorescence signatures from vegetation

NASA Astrophysics Data System (ADS)

Buschmann, C.

Vegetation emits fluorescence as part of the energy taken up by absorption %of solar radiation from UV to the visible. This fluorescence consists of light with low intensity (only few percents of the reflected light) emitted from the leaves. The fluorescence emission of a green leaf is characterized by four bands with maxima in the blue (440 nm), green (520 nm), red (690 nm) and far red (740 nm) spectral region. The intensity of fluorescence in the maxima of the emission spectrum varies depending on the following six basic parameters which must be taken into account for the interpretation of fluorescence signatures from vegetation: (a) content of the fluorophores (ferulic acid, chlorophyll a), (b) temperature of the leaf, (c) penetration of excitation light into the leaf, (d) emission of fluorescence from the leaf (re-absorption inside the leaf tissue), (e) photosynthetic activity of the leaf, (f) non-radiative decay (heat production) parallel to the fluorescence The ratios between the intensities of the maxima (F440/F690, F440/F520, F690/F740) are used as characteristic fluorescence parameter. The wide range of changes of these ratios caused by differences in the leaf tissue (aerial interspaces, variegated/homogeneous green leaves), various types of stress (UV, photoinhibition, sun exposure, heat, water deficiency, N-deficiency) and chemicals (inhibitors, fertilizers) can be explained by changes of the six basic parameters. It will be shown that the interpretation of the fluorescence signatures, in most cases, must be based on a complex consideration of more than one of the basic parameters.

Exciplex emission and decay of co-deposited 4,4',4″-tris[3-methylphenyl(phenyl)amino]triphenylamine:tris-[3-(3-pyridyl)mesityl]borane organic light-emitting devices with different electron transporting layer thicknesses

NASA Astrophysics Data System (ADS)

Huang, Qingyu; Zhao, Suling; Xu, Zheng; Fan, Xing; Shen, Chongyu; Yang, Qianqian

2014-04-01

Highly efficient fluorescence organic light-emitting diodes (OLEDs) based on the mixed 4,4',4″-tris[3-methylphenyl(phenyl)amino]triphenylamine:tris-[3-(3-pyridyl)mesityl]borane (1:1) system are reported. The electroluminescence due to the exciplex emission is red shifted when the thickness of the electron-transporting layer increases. The prepared OLEDs achieve a low turn-on voltage of 2.1 V, a high current efficiency of 36.79 cd/A, and a very high luminescence of 17 100 cd/m2, as well as a low efficiency roll-off. The current efficiency of the optimized OLED is maintained at more than 28.33 cd/A up to 10 000 cd m-2. The detailed recombination mechanism of the prepared OLEDs is investigated by the transient electroluminescence method. It is concluded that there are no contributions from trapped charges and annihilations of triplet-triplet excitons to the detected electroluminescence.

Fluorescent vancomycin and terephthalate comodified europium-doped layered double hydroxides nanoparticles: synthesis and application for bacteria labelling

NASA Astrophysics Data System (ADS)

Sun, Jianchao; Fan, Hai; Wang, Nan; Ai, Shiyun

2014-09-01

Vancomycin (Van)- and terephthalate (TA)-comodified europium-doped layered double hydroxides (Van-TA-Eu-LDHs) nanoparticles were successfully prepared by a two-step method, in which, TA acted as a sensitizer to enhance the fluorescent property and Van was modified on the surface of LDH to act as an affinity reagent to bacteria. The obtained products were characterized by X-ray diffraction, transmission electron microscope and fluorescent spectroscopy. The results demonstrated that the prepared Van- and TA-comodified europium-doped layered double hydroxides (Van-TA-Eu-LDHs) nanoparticles with diameter of 50 nm in size showed highly efficient fluorescent property. Furthermore, due to the high affinity of Van to bacteria, the prepared Van-TA-Eu-LDHs nanoparticles showed efficient bacteria labelling by fluorescent property. The prepared nanoparticles may have wide applications in the biological fields, such as biomolecular labelling and cell imaging.

Restoration of visual response in aged dystrophic RCS rats using AAV-mediated channelopsin-2 gene transfer.

PubMed

Tomita, Hiroshi; Sugano, Eriko; Yawo, Hiromu; Ishizuka, Toru; Isago, Hitomi; Narikawa, Satoko; Kügler, Sebastian; Tamai, Makoto

2007-08-01

To investigate whether the channelopsin-2 (Chop2) gene would restore visual responses in 10-month-old dystrophic Royal College of Surgeons (aged RCS; rdy/rdy) rats, the authors transferred the Chop2 gene into the retinal cells of aged RCS rats using the adenoassociated virus (AAV) vector. The N-terminal fragment (residues 1-315) of Chop2 was fused to a fluorescent protein, Venus, in frame at the end of the Chop2 coding fragment. The viral vector construct (AAV-Chop2V) for the expression of the Chop2V in the retina was made by subcloning into an adenoassociated virus vector, including the CAG promoter. To evaluate the expression profile of Chop2V in the retina, the rats were killed and the eyes were removed and fixed with 4% paraformaldehyde in 0.1 M phosphate-buffered saline. Retinal wholemount specimens and cryosections were made. Under anesthetized conditions, electrodes for the recording of visually evoked potentials (VEPs) were implanted onto the visual cortex in aged-RCS (rdy/rdy) rats. AAV-Chop2V vectors were then injected into the vitreous cavity of the left eyes. As a control, AAV-Venus vectors were applied to the right eyes. VEPs were evoked by the flash of a blue, white, or red light-emitting diode (LED) and were recorded from the visual cortex of the rats at various time points after the AAV vector injection. Chop2V fluorescence was predominantly observed in retinal ganglion cells (RGCs). Some fluorescence was observed in the inner nuclear layer and the inner plexiform layer neurites. A tendency of recovery was observed in the VEPs of aged RCS (rdy/rdy) rats after the AAV-Chop2V injection but not after the AAV-Venus injection. The visual response of AAV-Chop2V-injected aged RCS (rdy/rdy) rats was less sensitive to the blue LED flash than that of nondystrophic RCS (+/+) rats. The AAV-Chop2V-injected aged RCS (rdy/rdy) rats were insensitive to the red LED flash, which evoked a robust VEP in the RCS (+/+) rats. The visual response of aged RCS (rdy/rdy) rats was partially restored by transduction of the Chop2 gene through AAV into the inner retinal neurons, mainly RGCs. These results suggest that the transduction of Chop2 would provide a new strategy to treat some retinitis pigmentosa (RP) symptoms independent of their etiology.

Understanding Blue-to-Red Conversion in Monomeric Fluorescent Timers and Hydrolytic Degradation of Thier Chromophores

DOE Office of Scientific and Technical Information (OSTI.GOV)

Pletnev, Sergei; Subach, Fedor V.; Dauter, Zbigniew

2010-03-12

Fast-FT is a fluorescent timer (FT) engineered from DsRed-like fluorescent protein mCherry. Crystal structures of Fast-FT (chromophore Met66-Tyr67-Gly68) and its precursor with blocked blue-to-red conversion Blue102 (chromophore Leu66-Tyr67-Gly68) have been determined at the resolution of 1.15 {angstrom} and 1.81 {angstrom}, respectively. Structural data suggest that blue-to-red conversion, taking place in Fast-FT and in related FTs, is associated with the oxidation of C{alpha}2-C{beta}2 bond of Tyr67. Site directed mutagenesis revealed a crucial role of Arg70 and Tyr83 in the delayed oxidation of C{alpha}2-C{beta}2 bond, introducing the timing factor in maturation of the timer. Substitutions Ser217Ala and Ser217Cys in Fast-FT substantially slowmore » down formation of an intermediate blue chromophore but do not affect much blue-to-red conversion, whereas mutations Arg70Lys or Trp83Leu, having little effect on the blue chromophore formation rate, markedly accelerates formation of the red chromophore. The chromophore of FTs adopts a cis-conformation stabilized by a hydrogen bond between its phenolate oxygen and the side chain hydroxyl of Ser146. In Blue102, a bulky side chain of Ile146 precludes the chromophore from adopting a 'cis-like' conformation, blocking its blue-to-red conversion. Both Fast-FT and Blue102 structures revealed hydrolytic degradation of the chromophores. In Fast-FT, chromophore-forming Met66 residue is eliminated from the polypeptide chain, whereas Leu66 in Blue102 is cleaved out from the chromophore, decarboxylated and remains attached to the preceding Phe65. Hydrolysis of the chromophore competes with chromophore maturation and is driven by the same residues that participate in chromophore maturation.« less

trans- cis Isomerism and acylimine formation in DsRed chromophore models: Intrinsic rotation barriers

NASA Astrophysics Data System (ADS)

Olsen, Seth; Smith, Sean C.

2006-07-01

The chromophore of the red fluorescent protein DsRed contains an acylimine substituent to a GFP-like chromophore structure. The acylimine is formed from the trans peptide linkage between residues F65 and Q66 in immature DsRed, but has a cis configuration in the mature protein. The relationship between acylimine formation and trans- cis isomerization is unresolved. We have calculated bond rotation profiles for models of mature and immature DsRed chromophores using B3LYP DFT. The isomerization barrier is substantially reduced in acylimine-substituted models, providing prima facie evidence that acylimine formation precedes trans- cis isomerization in DsRed chromophores.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chekini, M.; Bierwagen, J.; Cunningham, A.

Localized surface plasmon resonances excited in metallic nanoparticles confine and enhance electromagnetic fields at the nanoscale. This is particularly pronounced in dimers made from two closely spaced nanoparticles. When quantum emitters, such as dyes, are placed in the gap of those dimers, their absorption and emission characteristics can be modified. Both processes have to be considered when aiming to enhance the fluorescence from the quantum emitters. This is particularly challenging for dimers, since the electromagnetic properties and the enhanced fluorescence sensitively depend on the distance between the nanoparticles. Here, we use a layer-by-layer method to precisely control the distances inmore » such systems. We consider a dye layer deposited on top of an array of gold nanoparticles or integrated into a central position of a double array of gold nanoparticles. We study the effect of the spatial arrangement and the average distance on the plasmon-enhanced fluorescence. We found a maximum of a 99-fold increase in the fluorescence intensity of the dye layer sandwiched between two gold nanoparticle arrays. The interaction of the dye layer with the plasmonic system also causes a spectral shift in the emission wavelengths and a shortening of the fluorescence life times. Our work paves the way for large-scale, high throughput, and low-cost self-assembled functionalized plasmonic systems that can be used as efficient light sources.« less

Uptake of Fluorescent Gentamicin by Peripheral Vestibular Cells after Systemic Administration

PubMed Central

Liu, Jianping; Kachelmeier, Allan; Dai, Chunfu; Li, Hongzhe; Steyger, Peter S.

2015-01-01

Objective In addition to cochleotoxicity, systemic aminoglycoside pharmacotherapy causes vestibulotoxicity resulting in imbalance and visual dysfunction. The underlying trafficking routes of systemically-administered aminoglycosides from the vasculature to the vestibular sensory hair cells are largely unknown. We investigated the trafficking of systemically-administered gentamicin into the peripheral vestibular system in C56Bl/6 mice using fluorescence-tagged gentamicin (gentamicin-Texas-Red, GTTR) imaged by scanning laser confocal microscopy to determine the cellular distribution and intensity of GTTR fluorescence in the three semicircular canal cristae, utricular, and saccular maculae at 5 time points over 4 hours. Results Low intensity GTTR fluorescence was detected at 0.5 hours as both discrete puncta and diffuse cytoplasmic fluorescence. The intensity of cytoplasmic fluorescence peaked at 3 hours, while punctate fluorescence was plateaued after 3 hours. At 0.5 and 1 hour, higher levels of diffuse GTTR fluorescence were present in transitional cells compared to hair cells and supporting cells. Sensory hair cells typically exhibited only diffuse cytoplasmic fluorescence at all time-points up to 4 hours in this study. In contrast, non-sensory cells rapidly exhibited both intense fluorescent puncta and weaker, diffuse fluorescence throughout the cytosol. The numbers and size of fluorescent puncta in dark cells and transitional cells increased over time. There is no preferential GTTR uptake by the five peripheral vestibular organs’ sensory cells. Control vestibular tissues exposed to Dulbecco’s phosphate-buffered saline or hydrolyzed Texas Red had negligible fluorescence. Conclusions All peripheral vestibular cells rapidly take up systemically-administered GTTR, reaching peak intensity 3 hours after injection. Sensory hair cells exhibited only diffuse fluorescence, while non-sensory cells displayed both diffuse and punctate fluorescence. Transitional cells may act as a primary pathway for trafficking of systemic GTTR from the vasculature to endolymph prior to entering hair cells. PMID:25793391

Multiparametric Flow Cytometry Using Near-Infrared Fluorescent Proteins Engineered from Bacterial Phytochromes

PubMed Central

Telford, William G.; Shcherbakova, Daria M.; Buschke, David; Hawley, Teresa S.; Verkhusha, Vladislav V.

2015-01-01

Engineering of fluorescent proteins (FPs) has followed a trend of achieving longer fluorescence wavelengths, with the ultimate goal of producing proteins with both excitation and emission in the near-infrared (NIR) region of the spectrum. Flow cytometers are now almost universally equipped with red lasers, and can now be equipped with NIR lasers as well. Most red-shifted FPs of the GFP-like family are maximally excited by orange lasers (590 to 610 nm) not commonly found on cytometers. This has changed with the development of the iRFP series of NIR FPs from the protein family of bacterial phytochromes. The shortest wavelength variants of this series, iRFP670 and iRFP682 showed maximal excitation with visible red lasers. The longer wavelength variants iRFP702, iRFP713 and iRFP720 could be optimally excited by NIR lasers ranging from 685 to 730 nm. Pairs of iRFPs could be detected simultaneously by using red and NIR lasers. Moreover, a novel spectral cytometry technique, which relies on spectral deconvolution rather than optical filters, allowed spectra of all five iRFPs to be analyzed simultaneously with no spectral overlap. Together, the combination of iRFPs with the advanced flow cytometry will allow to first image tissues expressing iRFPs deep in live animals and then quantify individual cell intensities and sort out the distinct primary cell subpopulations ex vivo. PMID:25811854

Improving brightness and photostability of green and red fluorescent proteins for live cell imaging and FRET reporting

PubMed Central

Bajar, Bryce T.; Wang, Emily S.; Lam, Amy J.; Kim, Bongjae B.; Jacobs, Conor L.; Howe, Elizabeth S.; Davidson, Michael W.; Lin, Michael Z.; Chu, Jun

2016-01-01

Many genetically encoded biosensors use Förster resonance energy transfer (FRET) to dynamically report biomolecular activities. While pairs of cyan and yellow fluorescent proteins (FPs) are most commonly used as FRET partner fluorophores, respectively, green and red FPs offer distinct advantages for FRET, such as greater spectral separation, less phototoxicity, and lower autofluorescence. We previously developed the green-red FRET pair Clover and mRuby2, which improves responsiveness in intramolecular FRET reporters with different designs. Here we report the engineering of brighter and more photostable variants, mClover3 and mRuby3. mClover3 improves photostability by 60% and mRuby3 by 200% over the previous generation of fluorophores. Notably, mRuby3 is also 35% brighter than mRuby2, making it both the brightest and most photostable monomeric red FP yet characterized. Furthermore, we developed a standardized methodology for assessing FP performance in mammalian cells as stand-alone markers and as FRET partners. We found that mClover3 or mRuby3 expression in mammalian cells provides the highest fluorescence signals of all jellyfish GFP or coral RFP derivatives, respectively. Finally, using mClover3 and mRuby3, we engineered an improved version of the CaMKIIα reporter Camuiα with a larger response amplitude. PMID:26879144

Multiparametric flow cytometry using near-infrared fluorescent proteins engineered from bacterial phytochromes.

PubMed

Telford, William G; Shcherbakova, Daria M; Buschke, David; Hawley, Teresa S; Verkhusha, Vladislav V

2015-01-01

Engineering of fluorescent proteins (FPs) has followed a trend of achieving longer fluorescence wavelengths, with the ultimate goal of producing proteins with both excitation and emission in the near-infrared (NIR) region of the spectrum. Flow cytometers are now almost universally equipped with red lasers, and can now be equipped with NIR lasers as well. Most red-shifted FPs of the GFP-like family are maximally excited by orange lasers (590 to 610 nm) not commonly found on cytometers. This has changed with the development of the iRFP series of NIR FPs from the protein family of bacterial phytochromes. The shortest wavelength variants of this series, iRFP670 and iRFP682 showed maximal excitation with visible red lasers. The longer wavelength variants iRFP702, iRFP713 and iRFP720 could be optimally excited by NIR lasers ranging from 685 to 730 nm. Pairs of iRFPs could be detected simultaneously by using red and NIR lasers. Moreover, a novel spectral cytometry technique, which relies on spectral deconvolution rather than optical filters, allowed spectra of all five iRFPs to be analyzed simultaneously with no spectral overlap. Together, the combination of iRFPs with the advanced flow cytometry will allow to first image tissues expressing iRFPs deep in live animals and then quantify individual cell intensities and sort out the distinct primary cell subpopulations ex vivo.

Real-time and quantitative fluorescent live-cell imaging with quadruplex-specific red-edge probe (G4-REP).

PubMed

Yang, Sunny Y; Amor, Souheila; Laguerre, Aurélien; Wong, Judy M Y; Monchaud, David

2017-05-01

The development of quadruplex-directed molecular diagnostic and therapy rely on mechanistic insights gained at both cellular and tissue levels by fluorescence imaging. This technique is based on fluorescent reporters that label cellular DNA and RNA quadruplexes to spatiotemporally address their complex cell biology. The photophysical characteristics of quadruplex probes usually dictate the modality of cell imaging by governing the selection of the light source (lamp, LED, laser), the optical light filters and the detection modality. Here, we report the characterizations of prototype from a new generation of quadruplex dye termed G4-REP (for quadruplex-specific red-edge probe) that provides fluorescence responses regardless of the excitation wavelength and modality (owing to the versatility gained through the red-edge effect), thus allowing for diverse applications and most imaging facilities. This is demonstrated by cell images (and associated quantifications) collected through confocal and multiphoton microscopy as well as through real-time live-cell imaging system over extended period, monitoring both non-cancerous and cancerous human cell lines. Our results promote a new way of designing versatile, efficient and convenient quadruplex-reporting dyes for tracking these higher-order nucleic acid structures in living human cells. This article is part of a Special Issue entitled "G-quadruplex" Guest Editor: Dr. Concetta Giancola and Dr. Daniela Montesarchio. Copyright © 2016 Elsevier B.V. All rights reserved.

Automated microscopy system for detection and genetic characterization of fetal nucleated red blood cells on slides

NASA Astrophysics Data System (ADS)

Ravkin, Ilya; Temov, Vladimir

1998-04-01

The detection and genetic analysis of fetal cells in maternal blood will permit noninvasive prenatal screening for genetic defects. Applied Imaging has developed and is currently evaluating a system for semiautomatic detection of fetal nucleated red blood cells on slides and acquisition of their DNA probe FISH images. The specimens are blood smears from pregnant women (9 - 16 weeks gestation) enriched for nucleated red blood cells (NRBC). The cells are identified by using labeled monoclonal antibodies directed to different types of hemoglobin chains (gamma, epsilon); the nuclei are stained with DAPI. The Applied Imaging system has been implemented with both Olympus BX and Nikon Eclipse series microscopes which were equipped with transmission and fluorescence optics. The system includes the following motorized components: stage, focus, transmission, and fluorescence filter wheels. A video camera with light integration (COHU 4910) permits low light imaging. The software capabilities include scanning, relocation, autofocusing, feature extraction, facilities for operator review, and data analysis. Detection of fetal NRBCs is achieved by employing a combination of brightfield and fluorescence images of nuclear and cytoplasmic markers. The brightfield and fluorescence images are all obtained with a single multi-bandpass dichroic mirror. A Z-stack of DNA probe FISH images is acquired by moving focus and switching excitation filters. This stack is combined to produce an enhanced image for presentation and spot counting.

Bright monomeric photoactivatable red fluorescent protein for two-color super-resolution sptPALM of live cells.

PubMed

Subach, Fedor V; Patterson, George H; Renz, Malte; Lippincott-Schwartz, Jennifer; Verkhusha, Vladislav V

2010-05-12

Rapidly emerging techniques of super-resolution single-molecule microscopy of living cells rely on the continued development of genetically encoded photoactivatable fluorescent proteins. On the basis of monomeric TagRFP, we have developed a photoactivatable TagRFP protein that is initially dark but becomes red fluorescent after violet light irradiation. Compared to other monomeric dark-to-red photoactivatable proteins including PAmCherry, PATagRFP has substantially higher molecular brightness, better pH stability, substantially less sensitivity to blue light, and better photostability in both ensemble and single-molecule modes. Spectroscopic analysis suggests that PATagRFP photoactivation is a two-step photochemical process involving sequential one-photon absorbance by two distinct chromophore forms. True monomeric behavior, absence of green fluorescence, and single-molecule performance in live cells make PATagRFP an excellent protein tag for two-color imaging techniques, including conventional diffraction-limited photoactivation microscopy, super-resolution photoactivated localization microscopy (PALM), and single particle tracking PALM (sptPALM) of living cells. Two-color sptPALM imaging was demonstrated using several PATagRFP tagged transmembrane proteins together with PAGFP-tagged clathrin light chain. Analysis of the resulting sptPALM images revealed that single-molecule transmembrane proteins, which are internalized into a cell via endocytosis, colocalize in space and time with plasma membrane domains enriched in clathrin light-chain molecules.

Transgenic rats with green, red, and blue fluorescence: powerful tools for bioimaging, cell trafficking, and differentiation

NASA Astrophysics Data System (ADS)

Murakami, Takashi; Kobayashi, Eiji

2005-04-01

The rat represents a perfect animal for broadening medical experiments, because its physiology has been well understood in the history of experimental animals. In addition, its larger body size takes enough advantage for surgical manipulation, compared to the mouse. Many rat models mimicking human diseases, therefore, have been used in a variety of biomedical studies including physiology, pharmacology, transplantation, and immunology. In an effort to create the specifically designed rats for biomedical research and regenerative medicine, we have developed the engineered rat system on the basis of transgenic technology and succeeded in establishing various transgenic rat strains. The transgenic rats with green fluorescent protein (GFP) were generated in the two different strains (Wistar and Lewis), in which GFP is driven under the chicken beta-actin promoter and cytomegalovirus enhancer (CAG promoter). Their GFP expression levels were different in each organ, but the Lewis line expressed GFP strongly and ubiquitously in most of the organs compared with that of Wistar. For red fluorescence, DsRed2 was transduced to the Wistar rats: one line specifically expresses DsRed2 in the liver under the mouse albumin promoter, another is designed for the Cre/LoxP system as the double reporter rat (the initial DsRed2 expression turns on GFP in the presence of Cre recombinase). LacZ-transgenic rats represent blue color, and LacZ is driven the CAG (DA) or ROSA26 promoter (Lewis). Our unique transgenic rats" system highlights the powerful performance for the elucidation of many cellular processes in regenerative medicine, leading to innovative medical treatments.

Electron Shuttling by Dissolved Humic Substances: Using Fluorescence Spectroscopy to Move Beyond the Laboratory to Natural Lakes, Streams and Groundwaters

NASA Astrophysics Data System (ADS)

McKnight, D. M.

2017-12-01

Humic substances are an important class of reactive chemical species in natural waters, and one important role is their capacity to as an electron acceptor and/or electron shuttle to ferric iron present as solid phase ferric oxides. Several lines of evidence point to quinone-like moieties being the main redox active moieties that can be used by microbes in respiration. Concomitantly, the humic fraction of dissolved organic mater (DOM) contains the dominant fluorophores in many natural waters. Examination of excitation emission matrices (EEMs) across redox gradients in diverse aquatic systems show that the EEMs are generally red-shifted under reducing conditions, such as anoxic bottom waters in lakes and hypoxic waters in riparian wetlands. Furthermore, there is striking similarity between the humic fluorophores that are resolved by statistical analysis and the fluorescence spectra of model quinone compounds, with the more reduced species having red-shifted fluorescence spectra. This apparent red-shift can be quantified based on the distribution of apparently "quinone-like", "semi-quinone-like" and "hydroquinone-like" fluorophores determined by the PARAFAC statistical analysis. Because fluorescence spectroscopy can be applied at ambient DOM concentrations for samples that have been maintained in an anoxic condition, fluorescence spectroscopy can provide insight into the role of humic electron shuttling in natural systems. Examples are presented demosntrating the changing EEMs in anoxic bottomwaters in a lake in the McMurdo Dry Valleys following a major flood event and the role of organic material in the mobilization of arsenic in shallow groundwater in South East Asia.

Production of transgenic cloned pigs expressing the far-red fluorescent protein monomeric Plum.

PubMed

Watanabe, Masahito; Kobayashi, Mirina; Nagaya, Masaki; Matsunari, Hitomi; Nakano, Kazuaki; Maehara, Miki; Hayashida, Gota; Takayanagi, Shuko; Sakai, Rieko; Umeyama, Kazuhiro; Watanabe, Nobuyuki; Onodera, Masafumi; Nagashima, Hiroshi

2015-01-01

Monomeric Plum (Plum), a far-red fluorescent protein with photostability and photopermeability, is potentially suitable for in vivo imaging and detection of fluorescence in body tissues. The aim of this study was to generate transgenic cloned pigs exhibiting systemic expression of Plum using somatic cell nuclear transfer (SCNT) technology. Nuclear donor cells for SCNT were obtained by introducing a Plum-expression vector driven by a combination of the cytomegalovirus early enhancer and chicken beta-actin promoter into porcine fetal fibroblasts (PFFs). The cleavage and blastocyst formation rates of reconstructed SCNT embryos were 81.0% (34/42) and 78.6% (33/42), respectively. At 36-37 days of gestation, three fetuses systemically expressing Plum were obtained from one recipient to which 103 SCNT embryos were transferred (3/103, 2.9%). For generation of offspring expressing Plum, rejuvenated PFFs were established from one cloned fetus and used as nuclear donor cells. Four cloned offspring and one stillborn cloned offspring were produced from one recipient to which 117 SCNT embryos were transferred (5/117, 4.3%). All offspring exhibited high levels of Plum fluorescence in blood cells, such as lymphocytes, monocytes and granulocytes. In addition, the skin, heart, kidney, pancreas, liver and spleen also exhibited Plum expression. These observations demonstrated that transfer of the Plum gene did not interfere with the development of porcine SCNT embryos and resulted in the successful generation of transgenic cloned pigs that systemically expressed Plum. This is the first report of the generation and characterization of transgenic cloned pigs expressing the far-red fluorescent protein Plum.

Red phosphorescent organic light-emitting diodes based on the simple structure.

PubMed

Seo, Ji Hyun; Lee, Seok Jae; Kim, Bo Young; Choi, Eun Young; Han, Wone Keun; Lee, Kum Hee; Yoon, Seung Soo; Kim, Young Kwan

2012-05-01

We demonstrated that the simple layered red phosphorescent organic light-emitting diodes (OLEDs) are possible to have high efficiency, low driving voltage, stable roll-off efficiency, and pure emission color without hole injection and transport layers. We fabricated the OLEDs with a structure of ITO/CBP doped with Ir(pq)2(acac)/BPhen/Liq/Al, where the doping concentration of red dopant, Ir(pq)2(acac), was varied from 4% to 20%. As a result, the quantum efficiencies of 13.4, 11.2, 16.7, 10.8 and 9.8% were observed in devices with doping concentrations of 4, 8, 12, 16 and 20%, respectively. Despite of absence of the hole injection and transport layers, these efficiencies are superior to efficiencies of device with hole transporting layer due to direct hole injection from anode to dopant in emission layer.

Light propagation analysis for fluorescence measurements of a molecular probe in the brain

NASA Astrophysics Data System (ADS)

Asai, Kota; Togashi, Takuya; Okada, Eiji

2017-04-01

Light propagation in the slab head model that consists of five types of tissues was calculated to estimate the fluorescent intensity emerged from a molecular probe in the brain by a Monte Carlo simulation. The thickness of the scalp, skull and cerebrospinal fluid layer was varied to analyze the influence of the thickness of the superficial tissues on the fluorescent intensity detected on the scalp surface. The fluorescent intensity is exponentially reduced with increasing the depth of the brain surface. The thickness of the cerebrospinal fluid layer more significantly affects the fluorescent intensity than that of the scalp and skull.

Near-infrared Spectroscopic Observations of Comet C/2013 R1 (Lovejoy) by WINERED: CN Red-system Band Emission

NASA Astrophysics Data System (ADS)

Shinnaka, Yoshiharu; Kawakita, Hideyo; Kondo, Sohei; Ikeda, Yuji; Kobayashi, Naoto; Hamano, Satoshi; Sameshima, Hiroaki; Fukue, Kei; Matsunaga, Noriyuki; Yasui, Chikako; Izumi, Natsuko; Mizumoto, Misaki; Otsubo, Shogo; Takenaka, Keiichi; Watase, Ayaka; Kawanishi, Takafumi; Nakanishi, Kenshi; Nakaoka, Tetsuya

2017-08-01

Although high-resolution spectra of the CN red-system band are considered useful in cometary sciences, e.g., in the study of isotopic ratios of carbon and nitrogen in cometary volatiles, there have been few reports to date due to the lack of high-resolution (R ≡ λ/Δλ > 20,000) spectrographs in the near-infrared region around ˜1 μm. Here, we present the high-resolution emission spectrum of the CN red-system band in comet C/2013 R1 (Lovejoy), acquired by the near-infrared high-resolution spectrograph WINERED mounted on the 1.3 m Araki telescope at the Koyama Astronomical Observatory, Kyoto, Japan. We applied our fluorescence excitation models for CN, based on modern spectroscopic studies, to the observed spectrum of comet C/2013 R1 (Lovejoy) to search for CN isotopologues (13C14N and 12C15N). We used a CN fluorescence excitation model involving both a “pure” fluorescence excitation model for the outer coma and a “fully collisional” fluorescence excitation model for the inner coma region. Our emission model could reproduce the observed 12C14N red-system band of comet C/2013 R1 (Lovejoy). The derived mixing ratio between the two excitation models was 0.94(+0.02/-0.03):0.06(+0.03/-0.02), corresponding to the radius of the collision-dominant region of ˜800-1600 km from the nucleus. No isotopologues were detected. The observed spectrum is consistent, within error, with previous estimates in comets of 12C/13C (˜90) and 14N/15N (˜150).

Cellular organization and spectral diversity of GFP-like proteins in live coral cells studied by single and multiphoton imaging and microspectroscopy

NASA Astrophysics Data System (ADS)

Salih, Anya; Cox, Guy C.; Larkum, Anthony W.

2003-07-01

Tissues of many marine invertebrates of class Anthozoa contain intensely fluorescent or brightly coloured pigments. These pigments belong to a family of photoactive proteins closely related to Green Fluorescent Protein (GFP), and their emissions range from blue to red wavelengths. The great diversity of these pigments has only recently been realised. To investigate the role of these proteins in corals, we have performed an in vivo fluorescent pigment (FP) spectral and cellular distribution analyses in live coral cells using single and multi-photon laser scanning imaging and microspectroscopy. These analyses revealed that even single colour corals contain spectroscopically heterogeneous pigment mixtures, with 2-5 major colour types in the same area of tissue. They were typically arranged in step-wise light emission energy gradients (e.g. blue, green, yellow, red). The successive overlapping emission-excitation spectral profiles of differently coloured FPs suggested that they were suited for sequential energy coupling. Traces of red FPs (emission = 570-660 nm) were present, even in non-red corals. We confirmed that radiative energy transfer could occur between separate granules of blue and green FPs and that energy transfer was inversely proportional to the square of the distance between them. Multi-photon micro-spectrofluorometric analysis gave significantly improved spectral resolution by restricting FP excitation to a single point in the focal plane of the sample. Pigment heterogeneity at small scales within granules suggested that fluorescence resonance energy transfer (FRET) might be occurring, and we confirmed that this was the case. Thus, energy transfer can take place both radiatively and by FRET, probably functioning in photoprotection by dissipation of excessive solar radiation.

Multiple-probe analysis of folding and unfolding pathways of human serum albumin. Evidence for a framework mechanism of folding.

PubMed

Santra, Manas Kumar; Banerjee, Abhijit; Krishnakumar, Shyam Sundar; Rahaman, Obaidur; Panda, Dulal

2004-05-01

The changes in the far-UV CD signal, intrinsic tryptophan fluorescence and bilirubin absorbance showed that the guanidine hydrochloride (GdnHCl)-induced unfolding of a multidomain protein, human serum albumin (HSA), followed a two-state process. However, using environment sensitive Nile red fluorescence, the unfolding and folding pathways of HSA were found to follow a three-state process and an intermediate was detected in the range 0.25-1.5 m GdnHCl. The intermediate state displayed 45% higher fluorescence intensity than that of the native state. The increase in the Nile red fluorescence was found to be due to an increase in the quantum yield of the HSA-bound Nile red. Low concentrations of GdnHCl neither altered the binding affinity of Nile red to HSA nor induced the aggregation of HSA. In addition, the secondary structure of HSA was not perturbed during the first unfolding transition (<1.5 m GdnHCl); however, the secondary structure was completely lost during the second transition. The data together showed that the half maximal loss of the tertiary structure occurred at a lower GdnHCl concentration than the loss of the secondary structure. Further kinetic studies of the refolding process of HSA using multiple spectroscopic techniques showed that the folding occurred in two phases, a burst phase followed by a slow phase. An intermediate with native-like secondary structure but only a partial tertiary structure was found to form in the burst phase of refolding. Then, the intermediate slowly folded into the native state. An analysis of the refolding data suggested that the folding of HSA could be best explained by the framework model.

Construction of Various γ34.5 Deleted Fluorescent-Expressing Oncolytic herpes Simplex type 1 (oHSV) for Generation and Isolation of HSV-Based Vectors

PubMed

Abdoli, Shahriyar; Roohvand, Farzin; Teimoori-Toolabi, Ladan; Shokrgozar, Mohammad Ali; Bahrololoumi, Mina; Azadmanesh, Kayhan

2017-07-01

Oncolytic herpes simplex virus (oHSV)-based vectors lacking γ34.5 gene, are considered as ideal templates to construct efficient vectors for (targeted) cancer gene therapy. Herein, we reported the construction of three single/dually-flourescence labeled and γ34.5-deleted, recombinant HSV-1 vectors for rapid generation and easy selection/isolation of different HSV-Based vectors. Generation of recombinant viruses was performed with conventional homologous recombination methods using green fluorescent protein (GFP) and BleCherry harboring shuttle vectors. Viruses were isolated by direct fluorescence observation and standard plaque purifying methods and confirmed by PCR and sequencing and flow cytometry. XTT and plaque assay titration were performed on Vero, U87MG, and T98 GBM cell lines. We generated three recombinant viruses, HSV-GFP, HSV-GR (Green-Red), and HSV-Red. The HSV-GFP showed two log higher titer (1010 PFU) than wild type (108 PFU). In contrast, HSV-GR and HSV-Red showed one log lower titer (107 PFU) than parental HSV. Cytotoxicity analysis showed that HSV-GR and HSV-Red can lyse target tumor cells at multiplicity of infection of 10 and 1 (P<0.001). Moreover, HSV-GFP showed higher infection potency (98%) in comparison with HSV-GR (82%). Our oHSVs provide a simple and an efficient platform for construction and rapid isolation of 2nd and 3rd generation oHSVs by replacing the inserted dyes with transgenes and also for rapid identification via fluorescence activated cell sorting. These vectors can also be used for tracing the efficacy of therapeutic agents on target cells, imaging of neural or tumoral cells in vitro/in vivo and as oncolytic agents in cancer therapy.

Photobleaching of red fluorescence in oral biofilms.

PubMed

Hope, C K; de Josselin de Jong, E; Field, M R T; Valappil, S P; Higham, S M

2011-04-01

Many species of oral bacteria can be induced to fluoresce due to the presence of endogenous porphyrins, a phenomenon that can be utilized to visualize and quantify dental plaque in the laboratory or clinical setting. However, an inevitable consequence of fluorescence is photobleaching, and the effects of this on longitudinal, quantitative analysis of dental plaque have yet to be ascertained. Filter membrane biofilms were grown from salivary inocula or single species (Prevotella nigrescens and Prevotella intermedia). The mature biofilms were then examined in a custom-made lighting rig comprising 405 nm light-emitting diodes capable of delivering 220 W/m(2) at the sample, an appropriate filter and a digital camera; a set-up analogous to quantitative light-induced fluorescence digital. Longitudinal sets of images were captured and processed to assess the degradation in red fluorescence over time. Photobleaching was observed in all instances. The highest rates of photobleaching were observed immediately after initiation of illumination, specifically during the first minute. Relative rates of photobleaching during the first minute of exposure were 19.17, 13.72 and 3.43 arbitrary units/min for P. nigrescens biofilms, microcosm biofilm and P. intermedia biofilms, respectively. Photobleaching could be problematic when making quantitative measurements of porphyrin fluorescence in situ. Reducing both light levels and exposure time, in combination with increased camera sensitivity, should be the default approach when undertaking analyses by quantitative light-induced fluorescence digital. © 2010 John Wiley & Sons A/S.

Evaluation of PpIX formation in Cervical Intraepithelial Neoplasia I (CIN) using widefield fluorescence images

NASA Astrophysics Data System (ADS)

Carbinatto, Fernanda M.; Inada, Natalia M.; Fortunato, Thereza C.; Lombardi, Welington; da Silva, Eduardo V.; Vollet Filho, José D.; Kurachi, Cristina; Pratavieira, Sebastião.; Bagnato, Vanderlei S.

2016-03-01

Optical techniques has been described as auxiliary technology for screening of neoplasia because shows the potential for tissues differentiation in real-time and it is a noninvasive detection and safe. However, only endogenous fluorophores presents the lesion may be insufficient and needed of the administration of the fluorophores synthesized, such as, precursor molecule of protoporphyrin IX (PpIX) induced by 5- aminolevulinic acid and your derivatives. Topical application of methylaminolevulinate (MAL), induces formation of the endogenous photosensitizer, PpIX in tissues where carcinogenesis has begun. The PpIX tend to accumulate in premalignant and malignant tissues and the illumination with light with appropriate wavelength beginning to excitation of PpIX fluorescence, which helps to localize PpIX-rich areas and identify potentially malignant tissues. The aim of the study is to evaluate the production of PpIX in the cervix with CIN I through of the fluorescence images captured after 1 hour of cream application. It was possible to visualize PpIX fluorescence in cervix and it was possible to observe the selectivity in fluorescence in squamous-columnar junction, which a pre-cancerous condition (CIN) and usually is localized. Through the image processing it was possible to quantify the increase of red fluorescence. For the CIN I the increase of red fluorescence was approximately of 4 times indicating a good PpIX formation.

[Discussion on diagenesis of Xilingang pluton-constrained by X-ray Fluorescence spectroscopy, plasma mass spectrometry and Raman spectroscopy].

PubMed

Tang, Yu-Kun; Chen, Guo-Neng; Zhang, Ke; Huang, Hai-Hua

2013-05-01

The results on Xilingang pluton, mainly consisting of red beds, granites containing numerous debris of red beds and granites, obtained by X-ray fluorescence spectroscopy, plasma mass spectrometry and Raman spectroscopy show: (1) Xilingang pluton from red beds, granites containing numerous debris of red beds to granites has obvious characteristics of decreasing silicon and alkali content, and rising ignition loss, dark mineral content and oxidation index; (2) Chondrite-normalized REE distribution curves and primitive mantle-normalized spider diagram for trace elements of redbed, granites containing numerous debris of red beds and granites have a good consistency, the distribution characteristics of elements are similar to Nanling transformation-type granite; (3) The value of Raman spectrogram characteristic peak of quartz crystal in Xilingang granite decreased from the center of quartz crystal, and FWHM is steady. According to the above, the authors believe that Xilingang granite formed was related to in-situ melting of red beds and underlying strata and magma consolidation. Volatile components were discharged continuously, and oxidation index decreased gradually in the melting process. In the process of diagenesis, the top of pluton tend to be an ongoing silicon and alkali increase, while TFeO and MgO continue to migrate to bottom, and crystallization environment is a relatively closed and steady system.

Broadband Fluorescence Enhancement with Self-Assembled Silver Nanoparticle Optical Antennas.

PubMed

Vietz, Carolin; Kaminska, Izabela; Sanz Paz, Maria; Tinnefeld, Philip; Acuna, Guillermo P

2017-05-23

Plasmonic structures are known to affect the fluorescence properties of dyes placed in close proximity. This effect has been exploited in combination with single-molecule techniques for several applications in the field of biosensing. Among these plasmonic structures, top-down zero-mode waveguides stand out due to their broadband capabilities. In contrast, optical antennas based on gold nanostructures exhibit fluorescence enhancement on a narrow fraction of the visible spectrum typically restricted to the red to near-infrared region. In this contribution, we exploit the DNA origami technique to self-assemble optical antennas based on large (80 nm) silver nanoparticles. We have studied the performance of these antennas with far- and near-field simulations and characterized them experimentally with single-molecule fluorescence measurements. We demonstrate that silver-based optical antennas can yield a fluorescence enhancement of more than 2 orders of magnitude throughout the visible spectral range for high intrinsic quantum yield dyes. Additionally, a comparison between the performance of gold and silver-based antennas is included. The results indicate that silver-based antennas strongly outperform their gold counterparts in the blue and green ranges and exhibit marginal differences in the red range. These characteristics render silver-based optical antennas ready for applications involving several fluorescently labeled species across the visible spectrum.

Fluorescent reversible regulation based on the interactions of topotecan hydrochloride, neutral red and quantum dots

NASA Astrophysics Data System (ADS)

Wang, Linlin; Shen, Yizhong; Liu, Shaopu; Yang, Jidong; Liang, Wanjun; Li, Dan; He, Youqiu

2015-02-01

The interactions of topotecan hydrochloride (THC), neutral red (NR) and thioglycolic acid (TGA) capped CdTe/CdS quantum dots (QDs) built a solid base for the controlling of the fluorescent reversible regulation of the system. This study was developed by means of ultraviolet-visible (UV-vis) absorption, fluorescence (FL), resonance Rayleigh scattering (RRS) spectroscopy and transmission electron microscopy (TEM). Corresponding experimental results revealed that the fluorescence of TGA-CdTe/CdS QDs could be effectively quenched by NR, while the RRS of the QDs enhanced gradually with the each increment of NR concentration. After the addition of THC, the strong covalent conjugation between NR and THC which was in carboxylate state enabled NR to be dissociated from the surface of TGA-CdTe/CdS QDs to form more stable complex with THC, thereby enhancing the fluorescence of the TGA-CdTe/CdS QDs-NR system. What is more, through analyzing the optical properties and experimental data of the reaction between TGA-CdTe/CdS QDs and NR, the possible reaction mechanism of the whole system was discussed. This combination of multiple spectroscopic techniques could contribute to the investigation for the fluorescent reversible regulation of QDs and a method could also be established to research the interactions between camptothecin drugs and dyes.

Method Of Signal Amplification In Multi-Chromophore Luminescence Sensors

DOEpatents

Levitsky, Igor A.; Krivoshlykov, Sergei G.

2004-02-03

A fluorescence-based method for highly sensitive and selective detection of analyte molecules is proposed. The method employs the energy transfer between two or more fluorescent chromophores in a carefully selected polymer matrix. In one preferred embodiment, signal amplification has been achieved in the fluorescent sensing of dimethyl methylphosphonate (DMMP) using two dyes, 3-aminofluoranthene (AM) and Nile Red (NR), in a hydrogen bond acidic polymer matrix. The selected polymer matrix quenches the fluorescence of both dyes and shifts dye emission and absorption spectra relative to more inert matrices. Upon DMMP sorption, the AM fluorescence shifts to the red at the same time the NR absorption shifts to the blue, resulting in better band overlap and increased energy transfer between chromophores. In another preferred embodiment, the sensitive material is incorporated into an optical fiber system enabling efficient excitation of the dye and collecting the fluorescent signal form the sensitive material on the remote end of the system. The proposed method can be applied to multichromophore luminescence sensor systems incorporating N-chromophores leading to N-fold signal amplification and improved selectivity. The method can be used in all applications where highly sensitive detection of basic gases, such as dimethyl methylphosphonate (DMMP), Sarin, Soman and other chemical warfare agents having basic properties, is required, including environmental monitoring, chemical industry and medicine.

Organic light-emitting device with a phosphor-sensitized fluorescent emission layer

DOEpatents

Forrest, Stephen [Ann Arbor, MI; Kanno, Hiroshi [Osaka, JP

2009-08-25

The present invention relates to organic light emitting devices (OLEDs), and more specifically to OLEDS that emit light using a combination of fluorescent emitters and phosphorescent emitters. The emissive region of the devices of the present invention comprise at least one phosphor-sensitized layer which has a combined emission from a phosphorescent emitter and a fluorescent emitter. In preferred embodiments, the invention relates to white-emitting OLEDS (WOLEDs).

Characterizing the Intrinsic Fluorescence Properties of Historical Painting Materials: The Case Study of a Sixteenth-Century Mesoamerican Manuscript.

PubMed

Pottier, Fabien; Michelin, Anne; Andraud, Christine; Goubard, Fabrice; Lavédrine, Bertrand

2018-04-01

Ultraviolet visible (UV-Vis) fluorescence spectroscopy is widely used to study polychrome objects and can help to identify the nature of certain materials when they present specific fluorescent properties. However, given the complexity of the stratified and heterogeneous materials under study, the characterization of an intrinsic fluorescence related to a given constituent (a pigment or a binder composing a paint layer for example) is not straightforward, and the recorded raw data need to be corrected for a number of effects that can influence the detected spectral distribution. The application of standard correction procedures to experimental fluorescence data gathered on the polychromatic surface of the Codex Borbonicus, a 16th-century Aztec manuscript, is described. The results are confronted to an alternate new methodology that is based on the hypothesis of transparent non-scattering paint layers. This second approach allows to establish more clearly the material origin of the detected emission and to discriminate apparent fluorescence (emitted by the substrate and transmitted through the paint layers) from actual intrinsic emission generated by the coloring materials under study. The results show that most of the various emission profiles detected in the paint layers of the manuscript actually originate from a unique fluorophore (composing the substrate) and should not be used to characterize the coloring materials.

Capillary waveguide optrodes: an approach to optical sensing in medical diagnostics

NASA Astrophysics Data System (ADS)

Lippitsch, Max E.; Draxler, Sonja; Kieslinger, Dietmar; Lehmann, Hartmut; Weigl, Bernhard H.

1996-07-01

Glass capillaries with a chemically sensitive coating on the inner surface are used as optical sensors for medical diagnostics. A capillary simultaneously serves as a sample compartment, a sensor element, and an inhomogeneous optical waveguide. Various detection schemes based on absorption, fluorescence intensity, or fluorescence lifetime are described. In absorption-based capillary waveguide optrodes the absorption in the sensor layer is analyte dependent; hence light transmission along the inhomogeneous waveguiding structure formed by the capillary wall and the sensing layer is a function of the analyte concentration. Similarly, in fluorescence-based capillary optrodes the fluorescence intensity or the fluorescence lifetime of an indicator dye fixed in the sensing layer is analyte dependent; thus the specific property of fluorescent light excited in the sensing layer and thereafter guided along the inhomogeneous waveguiding structure is a function of the analyte concentration. Both schemes are experimentally demonstrated, one with carbon dioxide as the analyte and the other one with oxygen. The device combines optical sensors with the standard glass capillaries usually applied to gather blood drops from fingertips, to yield a versatile diagnostic instrument, integrating the sample compartment, the optical sensor, and the light-collecting optics into a single piece. This ensures enhanced sensor performance as well as improved handling compared with other sensors. waveguide, blood gases, medical diagnostics.

Two colorimetric and ratiometric fluorescence probes for hydrogen sulfide based on AIE strategy of α-cyanostilbenes

NASA Astrophysics Data System (ADS)

Zhao, Baoying; Yang, Binsheng; Hu, Xiangquan; Liu, Bin

2018-06-01

Aggregation-induced emission (AIE) active fluorescent probes have attracted great potential in biological sensors. In this paper two cyanostilbene based fluorescence chemoprobe Cya-NO2 (1) and Cya-N3 (2) were developed and evaluated for the selective and sensitive detection of hydrogen sulfide (H2S). Both of these probes behave aggression-induced emission (AIE) activity which fluoresces in the red region with a large Stokes shift. They exhibit rapid response to H2S with enormous colorimetric and ratiometric fluorescent changes. They are readily employed for assessing intracellular H2S levels.

The consistent difference in red fluorescence in fishes across a 15 m depth gradient is triggered by ambient brightness, not by ambient spectrum.

PubMed

Harant, Ulrike Katharina; Michiels, Nicolaas Karel; Anthes, Nils; Meadows, Melissa Grace

2016-02-17

Organisms adapt to fluctuations or gradients in their environment by means of genetic change or phenotypic plasticity. Consistent adaptation across small spatial scales measured in meters, however, has rarely been reported. We recently found significant variation in fluorescence brightness in six benthic marine fish species across a 15 m depth gradient. Here, we investigate whether this can be explained by phenotypic plasticity alone, using the triplefin Tripterygion delaisi as a model species. In two separate experiments, we measure change in red fluorescent brightness to spectral composition and ambient brightness, two central parameters of the visual environment that change rapidly with depth. Changing the ambient spectra simulating light at -5 or -20 m depth generated no detectable changes in mean fluorescence brightness after 4-6 weeks. In contrast, a reduction in ambient brightness generated a significant and reversible increase in mean fluorescence, most of this within the first week. Although individuals can quickly up- and down-regulate their fluorescence around this mean value using melanosome aggregation and dispersal, we demonstrate that this range around the mean remained unaffected by either treatment. We show that the positive association between fluorescence and depth observed in the field can be fully explained by ambient light brightness, with no detectable additional effect of spectral composition. We propose that this change is achieved by adjusting the ratio of melanophores and fluorescent iridophores in the iris.

Electroluminescence and Photoluminescence from a Fluorescent Cobalt Porphyrin Grafted on Graphene Oxide

NASA Astrophysics Data System (ADS)

Janghouri, Mohammad

2017-10-01

A new graphene oxide-cobalt porphyrin (GO-CoTPP) hybrid material has been used as an emissive layer in organic light-emitting diodes (OLEDs). Devices with fundamental structure of indium-doped tin oxide (ITO)/poly(3,4-ethylenedioxythiophene):poly(styrenesulfonate) (PEDOT:PSS, 45 nm)/polyvinylcarbazole (PVK):2-(4-biphenyl)-5-(4- t-butylphenyl)-1,3,4-oxadiazole (PBD):GO-CoTPP (70 nm)/1,3,5-tris( N-phenylbenzimidazol-2-yl)-benzene (TPBI, 20 nm)/Al (150 nm) were fabricated. A red electroluminescence (EL) was obtained from thin-film PVK:PBD:CoTPP at 70 nm thickness. When CoTPP was covalently grafted on graphene oxide (GO) sheets, near-white EL was obtained. The white emission, which was composed of bluish green and red, is attributed to electroplex formation at the GO-CoTPP/PBD interface. Such electroplex emission between electrons and holes is a reason for the low turn-on voltage of the GO-CoTPP-based OLED. Maximum luminance efficiency of 1.43 cd/A with Commission International de l'Eclairage coordinates of 0.33 and 0.40 was achieved at current of 0.02 mA and voltage of 14 V.

[Characteristics of soil moisture in artificial impermeable layers].

PubMed

Suo, Gai-Di; Xie, Yong-Sheng; Tian, Fei; Chuai, Jun-Feng; Jing, Min-Xiao

2014-09-01

For the problem of low water and fertilizer use efficiency caused by nitrate nitrogen lea- ching into deep soil layer and soil desiccation in dryland apple orchard, characteristics of soil moisture were investigated by means of hand tamping in order to find a new approach in improving the water and fertilizer use efficiency in the apple orchard. Two artificial impermeable layers of red clay and dark loessial soil were built in soil, with a thickness of 3 or 5 cm. Results showed that artificial impermeable layers with the two different thicknesses were effective in reducing or blocking water infiltration into soil and had higher seepage controlling efficiency. Seepage controlling efficiency for the red clay impermeable layer was better than that for the dark loessial soil impermeable layer. Among all the treatments, the red clay impermeable layer of 5 cm thickness had the highest bulk density, the lowest initial infiltration rate (0.033 mm · min(-1)) and stable infiltration rate (0.018 mm · min(-1)) among all treatments. After dry-wet alternation in summer and freezing-thawing cycle in winter, its physiochemical properties changed little. Increase in years did not affect stable infiltration rate of soil water. The red clay impermeable layer of 5 cm thickness could effectively increase soil moisture content in upper soil layer which was conducive to raise the water and nutrient use efficiency. The approach could be applied to the apple production of dryland orchard.

The effect of a charge control layer on the electroluminescent characteristic of blue and white organic light-emitting diodes.

PubMed

Lee, Dong Hyung; Lee, Seok Jae; Koo, Ja-Ryong; Lee, Ho Won; Shin, Hyun Su; Lee, Song Eun; Kim, Woo Young; Lee, Kum Hee; Yoon, Seung Soo; Kim, Young Kwan

2014-08-01

We investigated blue fluorescent organic light-emitting diode (OLED) with a charge control layer (CCL) to produce high efficiency and improve the half-decay lifetime. Three types of devices (device A, B, and C) were fabricated following the number of CCLs within the emitting layer (EML), maintaining the thickness of whole EML. The CCL and host material, 2-methyl-9,10-di(2-naphthyl)anthracene, which has a bipolar property, was able to control the carrier movement with ease inside the EML. Device B demonstrated a maximum luminous efficiency (LE) and external quantum efficiency (EQE) of 9.19 cd/A and 5.78%, respectively. It also showed that the enhancement of the half-decay lifetime, measured at an initial luminance of 1,000 cd/m2, was 1.5 times longer than that of the conventional structure. A hybrid white OLED (WOLED) was also fabricated using a phosphorescent red emitter, bis(2-phenylquinoline)-acetylacetonate iridium III doped in 4,4'-N,N'-dicarbazolyl-biphenyl. The property of the hybrid WOLED with CCL showed a maximum LE and an EQE of 13.46 cd/A and 8.32%, respectively. It also showed white emission with Commission International de L'Éclairage coordinates of (x = 0.41, y = 0.33) at 10 V.

Curcumin as an amyloid-indicator dye in E. coli.

PubMed

McCrate, Oscar A; Zhou, Xiaoxue; Cegelski, Lynette

2013-05-14

We have demonstrated that curcumin is an amyloid-specific dye in E. coli. Curcumin binds to curliated whole cells and to isolated curli amyloid fibers. Similar to Congo red, curcumin exhibits a red-shift in absorbance and a significant increase in fluorescence upon binding to isolated curli.

Curcumin as an Amyloid-indicator Dye in E. coli †

PubMed Central

McCrate, Oscar A.; Zhou, Xiaoxue; Cegelski, Lynette

2013-01-01

We have demonstrated that curcumin is an amyloid-specific dye in E. coli. Curcumin binds to curliated whole cells and to isolated curli amyloid fibers. Similar to Congo red, curcumin exhibits a red-shift in absorbance and a significant increase in fluorescence upon binding to isolated curli. PMID:23287899

QUANTITATIVE FLUORESCENCE OF 5-FU-TREATED FETAL RAT LIMBS USING CONFOCAL LASER SCANNING MICROSCOPY AND LYSOTRACKER RED

EPA Science Inventory

Background: LysoTracker Red (LT) is a paraformaldehyde fixable probe that concentrates into acidic compartments of cells and tissues. After cell death a high level of lysosomal activity (acidic enzyme) is expressed resulting from phagocytosis of apoptotic bodies by neighboring ce...

Deeper Insight into Fluorescence--Excitation of Molecules by Light

NASA Astrophysics Data System (ADS)

Wahab, M. Farooq; Gore, Gordon R.

2013-05-01

In a recent issue of TPT, Gordon Gore made interesting observations about the red or yellow fluorescence when laser beams are passed through olive oil. With the excellent visuals contained in that article, we present a pictorial explanation of the questions that were raised in Ref 1.

NMDA Receptor Activity in Circulating Red Blood Cells: Methods of Detection.

PubMed

Makhro, Asya; Kaestner, Lars; Bogdanova, Anna

2017-01-01

Abundance and activity of N-methyl-D-aspartate (NMDA) in circulating red blood cells contributes to the maintenance of intracellular Ca 2+ in these cells and, by doing that, controls red cell volume, membrane stability, and O 2 carrying capacity. Detection of the NMDA receptor activity in red blood cells is challenging as the number of its copies is low and shows substantial cell-to-cell heterogeneity. Receptor abundance is reliably assessed using the radiolabeled antagonist ([ 3 H]MK-801) binding technique. Uptake of Ca 2+ following the NMDA receptor activation is detected in cells loaded with Ca 2+ -sensitive fluorescent dye Fluo-4 AM. Both microfluorescence live-cell imaging and flow cytometry may be used for fluorescence intensity detection. Automated patch clamp is currently used for recording of electric currents triggered by the stimulation of the NMDA receptor. These currents are mediated by the Ca 2+ -sensitive K + (Gardos) channels that open upon Ca 2+ uptake via the active NMDA receptor. Furthermore, K + flux through the Gardos channels induced by the NMDA receptor stimulation in red blood cells may be detected using unidirectional K + ( 86 Rb + ) influx.

Depth-resolved fluorescence of human ectocervical tissue

NASA Astrophysics Data System (ADS)

Wu, Yicong; Xi, Peng; Cheung, Tak-Hong; Yim, So Fan; Yu, Mei-Yung; Qu, Jianan Y.

2005-04-01

The depth-resolved autofluorescence of normal and dysplastic human ectocervical tissue within 120um depth were investigated utilizing a portable confocal fluorescence spectroscopy with the excitations at 355nm and 457nm. From the topmost keratinizing layer of all ectocervical tissue samples, strong keratin fluorescence with the spectral characteristics similar to collagen was observed, which created serious interference in seeking the correlation between tissue fluorescence and tissue pathology. While from the underlying non-keratinizing epithelial layer, the measured NADH fluorescence induced by 355nm excitation and FAD fluorescence induced by 457nm excitation were strongly correlated to the tissue pathology. The ratios between NADH over FAD fluorescence increased statistically in the CIN epithelial relative to the normal and HPV epithelia, which indicated increased metabolic activity in precancerous tissue. This study demonstrates that the depth-resolved fluorescence spectroscopy can reveal fine structural information on epithelial tissue and potentially provide more accurate diagnostic information for determining tissue pathology.

Spectral properties of the association nanoparticle system of ciprofloxacin-phloxine and its application to fluorescence analysis.

PubMed

Zou, Jieming; Jiang, Zhiliang; Wang, Lisheng; Li, Tingsheng; Liu, Qinye

2004-06-01

There is a fluorescence peak at 570 nm, and a maximum absorption peak at 560 nm for phloxine (PHLO) in a pH 7 water solution. Under these conditions, the ciprofloxacin cation (CPFX+) and PHLO- combine into hydrophobic CPFX-PHLO association molecule by means of static gravitation. There are stronger van der Waals forces and hydrophobic forces among the CPFX-PHLO molecules. Thus, they aggregate automatically to the (CPFX-PHLO)n association nanoparticle in red-violet color. That was characterized by scan electron microscopy (SEM), hyperfiltration and dialysis tests. In 0.04 M HCl, the red-violet nanoparticles exhibited a Rayleigh scattering peak at 470 nm, a resonance scattering peak at 580 nm, a maximum absorption wavelength at 565 nm, and a fluorescence peak at 450 nm. The fluorescence analytical conditions of CPFX have been considered. The CPFX concentration in the range of 1.0 x 10(-6)-4.0 x 10(-5) M is linear to the fluorescence intensity, F450nm. The detection limit was achieved at 4.0 x 10(-7) M CPFX. The CPFX in real samples was determined with satisfactory results.

Post-mortem re-cloning of a transgenic red fluorescent protein dog.

PubMed

Hong, So Gun; Koo, Ok Jae; Oh, Hyun Ju; Park, Jung Eun; Kim, Minjung; Kim, Geon-A; Park, Eun Jung; Jang, Goo; Lee, Byeong-Chun

2011-12-01

Recently, the world's first transgenic dogs were produced by somatic cell nuclear transfer. However, cellular senescence is a major limiting factor for producing more advanced transgenic dogs. To overcome this obstacle, we rejuvenated transgenic cells using a re-cloning technique. Fibroblasts from post-mortem red fluorescent protein (RFP) dog were reconstructed with in vivo matured oocytes and transferred into 10 surrogate dogs. One puppy was produced and confirmed as a re-cloned dog. Although the puppy was lost during birth, we successfully established a rejuvenated fibroblast cell line from this animal. The cell line was found to stably express RFP and is ready for additional genetic modification.

RNA "traffic lights": an analytical tool to monitor siRNA integrity.

PubMed

Holzhauser, Carolin; Liebl, Renate; Goepferich, Achim; Wagenknecht, Hans-Achim; Breunig, Miriam

2013-05-17

The combination of thiazole orange and thiazole red as an internal energy transfer-based fluorophore pair in oligonucleotides provides an outstanding analytical tool to follow DNA/RNA hybridization through a distinct fluorescence color change from red to green. Herein, we demonstrate that this concept can be applied to small interfering RNA (siRNA) to monitor RNA integrity in living cells in real time with a remarkable dynamic range and excellent contrast ratios in cellular media. Furthermore, we show that our siRNA-sensors still possess their gene silencing function toward the knockdown of enhanced green fluorescent protein in CHO-K1 cells.

Detection and characterization of stomach cancer and atrophic gastritis with fluorescence and Raman spectroscopy

NASA Astrophysics Data System (ADS)

Li, Xiaozhou; Lin, Junxiu; Jia, Chunde; Wang, Rong

2003-12-01

In this paper, we attempt to find a valid method to distinguish gastric cancer and atrophic gastritis. Auto-fluorescence and Raman spectroscopy of laser induced (514.5 nm and 488.0 nm) was measured. The serum spectrum is different between normal and cancer. Average value of diagnosis parameter for normal serum, red shift is less than 12 nm and Raman relative intensity of peak C by 514.5 nm excited is stronger than that of 488.0 nm. To gastric cancer, its red shift of average is bigger than 12 nm and relative intensity of Raman peak C by 514.5 nm excited is weaker than that by 488.0 nm. To atrophic gastritis, the distribution state of Raman peaks is similar with normal serum and auto-fluorescence spectrum's shape is similar to that of gastric cancer. Its average Raman peak red shift is bigger than 12 nm and the relative intensity of peak C by 514.5 excited is stronger than that of by 488.0. We considered it as a criterion and got an accuracy of 85.6% for diagnosis of gastric cancer compared with the result of clinical diagnosis.

18F-positron-emitting/fluorescent labeled erythrocytes allow imaging of internal hemorrhage in a murine intracranial hemorrhage model

PubMed Central

Wang, Ye; An, Fei-Fei; Chan, Mark; Friedman, Beth; Rodriguez, Erik A; Tsien, Roger Y; Aras, Omer

2017-01-01

An agent for visualizing cells by positron emission tomography is described and used to label red blood cells. The labeled red blood cells are injected systemically so that intracranial hemorrhage can be visualized by positron emission tomography (PET). Red blood cells are labeled with 0.3 µg of a positron-emitting, fluorescent multimodal imaging probe, and used to non-invasively image cryolesion induced intracranial hemorrhage in a murine model (BALB/c, 2.36 × 108 cells, 100 µCi, <4 mm hemorrhage). Intracranial hemorrhage is confirmed by histology, fluorescence, bright-field, and PET ex vivo imaging. The low required activity, minimal mass, and high resolution of this technique make this strategy an attractive alternative for imaging intracranial hemorrhage. PET is one solution to a spectrum of issues that complicate single photon emission computed tomography (SPECT). For this reason, this application serves as a PET alternative to [99mTc]-agents, and SPECT technology that is used in 2 million annual medical procedures. PET contrast is also superior to gadolinium and iodide contrast angiography for its lack of clinical contraindications. PMID:28054494

Combined thioflavin T-Congo red fluorescence assay for amyloid fibril detection

NASA Astrophysics Data System (ADS)

Girych, Mykhailo; Gorbenko, Galyna; Maliyov, Ivan; Trusova, Valeriya; Mizuguchi, Chiharu; Saito, Hiroyuki; Kinnunen, Paavo

2016-09-01

Fluorescence represents one of the most powerful tools for the detection and structural characterization of the pathogenic protein aggregates, amyloid fibrils. The traditional approaches to the identification and quantification of amyloid fibrils are based on monitoring the fluorescence changes of the benzothiazole dye thioflavin T (ThT) and absorbance changes of the azo dye Congo red (CR). In routine screening it is usually sufficient to perform only the ThT and CR assays, but both of them, when used separately, could give false results. Moreover, fibrillization kinetics can be measured only by ThT fluorescence, while the characteristic absorption spectra and birefringence of CR represent more rigid criteria for the presence of amyloid fibrils. Therefore, it seemed reasonable to use both these dyes simultaneously, combining the advantages of each technique. To this end, we undertook a detailed analysis of the fluorescence spectral behavior of these unique amyloid tracers upon their binding to amyloid fibrils from lysozyme, insulin and an N-terminal fragment of apolipoprotein A-I with Iowa mutation. The fluorescence measurements revealed several criteria for distinguishing between fibrillar and monomeric protein states: (i) a common drastic increase in ThT fluorescence intensity; (ii) a sharp decrease in ThT fluorescence upon addition of CR; (iii) an appearance of the maximum at 535-540 nm in the CR excitation spectra; (iv) increase in CR fluorescence intensity at 610 nm. Based on these findings we designed a novel combined ThT-CR fluorescence assay for amyloid identification. Such an approach not only strengthens the reliability of the ThT assay, but also provides new opportunities for structural characterization of amyloid fibrils.

[The transfection and expression of IL-1ra gene to the rabbit cornea in situ via cation polymer mediation].

PubMed

Yuan, Jin; Chen, Jia-qi; Zhou, Shi-you; Liu, Zu-guo; Wang, Zhi-chong; Gu, Jian-jun

2006-08-01

To investigate the efficiency and safety of transfection of PEGFP-IL-1ra plasmid via cation polymer mediation (poly-ethylenimine, PEI) by injection into the corneal stroma. Human IL-1ra cDNA fragments were cloned by RT-PCR. Plasmid PEGFP-hIL-1ra recombinants were constructed and transferred into corneal endothelial cells (CEC) via cation polymer mediation. Expression of IL-1ra mRNA and IL-1ra was detected by green fluorescent protein (GFP) and Western-blotting. In the experiment group, 20 microl preparation containing 10 microg plasmid PEGFP-hIL-1ra recombinants and PEI-in-vivo was injected into the corneal stroma of Wistar rats (n = 30). Equivalent PEI-in-vivo solution was injected into another 15 corneas as the controls. Corneas were harvested at different time points (day 1, 3, 6, 14 and 21) after injection. The changes of tissue structure and function after IL-1ra in situ transfection were studied by HE staining, transmission electron microscopy, trypan blue-alizarin red staining and immunohistochemistry. The location and intensity of IL-1ra-GFP fusion protein expression were monitored by fluorescence microscopy. The size of the RT-PCR product of hIL-1ra fragments was approximately 500 bp in agarose gel electrophoresis. Restrictive enzyme digestion analysis of PstI, BamHI and DNA sequence analysis showed that expression of plasmid PEGFP-hIL-1ra recombinants had been constructed successfully. Twelve hours after the transfection of PEGFP-hIL-1ra, GFP fluorescence was detected in 10% - 15% endothelial cells. IL-1ra protein (RMW: 44,000) was detected by Western-blotting. In PEGFP-hIL-1ra treated group, fluorescence was appeared at day 1 in cornea basal epithelial cells, peaked at day 6 in whole cornea, began to weaken at day 14, and only weak fluorescence remained in cornea epithelial cells at day 21. No fluorescence appeared in the control group. No significant pathologic changes could be found in HE stained cornea tissues in both transfected group and the controls. p63 immunocytochemical staining in cornea epithelium was positive in both groups. Trypan blue-alizarin red staining confirmed that there was no damage in cornea endothelial cells. IL-1ra-GFP granules could be found by transmission electron microscope in every layer of cornea in the transfected group, but none in the controls. There was no impairment in the ultrastructure of cells in both groups. By direct injection of PEGFP-hIL-1ra into corneal stroma and mediated by cation polymer, IL-1ra genes could be transferred and expressed in corneal tissue efficiently and safely, and might provide a novel technique of gene transfection to cornea in situ.