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.

Hybrid fluorescence and electron cryo-microscopy for simultaneous electron and photon imaging.

PubMed

Iijima, Hirofumi; Fukuda, Yoshiyuki; Arai, Yoshihiro; Terakawa, Susumu; Yamamoto, Naoki; Nagayama, Kuniaki

2014-01-01

Integration of fluorescence light and transmission electron microscopy into the same device would represent an important advance in correlative microscopy, which traditionally involves two separate microscopes for imaging. To achieve such integration, the primary technical challenge that must be solved regards how to arrange two objective lenses used for light and electron microscopy in such a manner that they can properly focus on a single specimen. To address this issue, both lateral displacement of the specimen between two lenses and specimen rotation have been proposed. Such movement of the specimen allows sequential collection of two kinds of microscopic images of a single target, but prevents simultaneous imaging. This shortcoming has been made up by using a simple optical device, a reflection mirror. Here, we present an approach toward the versatile integration of fluorescence and electron microscopy for simultaneous imaging. The potential of simultaneous hybrid microscopy was demonstrated by fluorescence and electron sequential imaging of a fluorescent protein expressed in cells and cathodoluminescence imaging of fluorescent beads. Copyright © 2013 Elsevier Inc. All rights reserved.

Evolution of molecular weight and fluorescence of effluent organic matter (EfOM) during oxidation processes revealed by advanced spectrographic and chromatographic tools.

PubMed

Chen, Zhiqiang; Li, Mo; Wen, Qinxue; Ren, Nanqi

2017-11-01

Effluent organic matter (EfOM) is an emerging concern to receiving aquatic environment due to its refractory property. The degradation of EfOM in ozonation and other two advanced oxidation processes (AOPs), UV/H 2 O 2 and UV/persulfate (PS), was investigated in this study. Fluorescence spectra coupled with parallel factor analysis (PARAFAC) and two-dimensional correlation gel permeation chromatography (2D-GPC) were used to track the evolution of EfOM during each oxidation process. Results showed that the degradation of EfOM indicated by dissolved organic carbon (DOC), UV 254 and fluorescence components, fitted well with pseudo-first-order kinetic model during the oxidation processes. Ozonation showed higher degradation efficiency than AOPs, while UV/PS was more effective than UV/H 2 O 2 with equimolar oxidants dosage. Ozone and SO· 4 - were more reactive with terrestrial humic-like substances, while hydroxyl radical preferentially reacted with protein-like substances. Organic molecules with higher molecular weight (MW) were susceptible to ozone or radicals. Ozonation could transform higher MW (MW of 3510 and 575) organic matters into lower MW organic matters (MW of 294), while reductions of all the organics were observed in both AOPs. Due to the higher reaction rates between ozone and EfOM, ozonation maybe serve as a pre-treatment for AOPs to reduce the radical and energy consumption and improve mineralization of EfOM by AOPs. The decline in DOC, UV 254 , fluorescence and reduction in oxidants increased with the increase of oxidants dosage, and linear correlations among them were found during the ozonation and AOPs. Copyright © 2017. Published by Elsevier Ltd.

Single-molecule spectroscopic methods.

PubMed

Haustein, Elke; Schwille, Petra

2004-10-01

Being praised for the mere fact of enabling the detection of individual fluorophores a dozen years ago, single-molecule techniques nowadays represent standard methods for the elucidation of the structural rearrangements of biologically relevant macromolecules. Single-molecule-sensitive techniques, such as fluorescence correlation spectroscopy, allow real-time access to a multitude of molecular parameters (e.g. diffusion coefficients, concentration and molecular interactions). As a result of various recent advances, this technique shows promise even for intracellular applications. Fluorescence imaging can reveal the spatial localization of fluorophores on nanometer length scales, whereas fluorescence resonance energy transfer supports a wide range of different applications, including real-time monitoring of conformational rearrangements (as in protein folding). Still in their infancy, single-molecule spectroscopic methods thus provide unprecedented insights into basic molecular mechanisms. Copyright 2004 Elsevier Ltd.

Inexpensive electronics and software for photon statistics and correlation spectroscopy.

PubMed

Gamari, Benjamin D; Zhang, Dianwen; Buckman, Richard E; Milas, Peker; Denker, John S; Chen, Hui; Li, Hongmin; Goldner, Lori S

2014-07-01

Single-molecule-sensitive microscopy and spectroscopy are transforming biophysics and materials science laboratories. Techniques such as fluorescence correlation spectroscopy (FCS) and single-molecule sensitive fluorescence resonance energy transfer (FRET) are now commonly available in research laboratories but are as yet infrequently available in teaching laboratories. We describe inexpensive electronics and open-source software that bridges this gap, making state-of-the-art research capabilities accessible to undergraduates interested in biophysics. We include a discussion of the intensity correlation function relevant to FCS and how it can be determined from photon arrival times. We demonstrate the system with a measurement of the hydrodynamic radius of a protein using FCS that is suitable for the undergraduate teaching laboratory. The FPGA-based electronics, which are easy to construct, are suitable for more advanced measurements as well, and several applications are described. As implemented, the system has 8 ns timing resolution, can control up to four laser sources, and can collect information from as many as four photon-counting detectors.

Inexpensive electronics and software for photon statistics and correlation spectroscopy

PubMed Central

Gamari, Benjamin D.; Zhang, Dianwen; Buckman, Richard E.; Milas, Peker; Denker, John S.; Chen, Hui; Li, Hongmin; Goldner, Lori S.

2016-01-01

Single-molecule-sensitive microscopy and spectroscopy are transforming biophysics and materials science laboratories. Techniques such as fluorescence correlation spectroscopy (FCS) and single-molecule sensitive fluorescence resonance energy transfer (FRET) are now commonly available in research laboratories but are as yet infrequently available in teaching laboratories. We describe inexpensive electronics and open-source software that bridges this gap, making state-of-the-art research capabilities accessible to undergraduates interested in biophysics. We include a discussion of the intensity correlation function relevant to FCS and how it can be determined from photon arrival times. We demonstrate the system with a measurement of the hydrodynamic radius of a protein using FCS that is suitable for the undergraduate teaching laboratory. The FPGA-based electronics, which are easy to construct, are suitable for more advanced measurements as well, and several applications are described. As implemented, the system has 8 ns timing resolution, can control up to four laser sources, and can collect information from as many as four photon-counting detectors. PMID:26924846

Advancements of two dimensional correlation spectroscopy in protein researches

NASA Astrophysics Data System (ADS)

Tao, Yanchun; Wu, Yuqing; Zhang, Liping

2018-05-01

The developments of two-dimensional correlation spectroscopy (2DCOS) applications in protein studies are discussed, especially for the past two decades. The powerful utilities of 2DCOS combined with various analytical techniques in protein studies are summarized. The emphasis is on the vibration spectroscopic techniques including IR, NIR, Raman and optical activity (ROA), as well as vibration circular dichroism (VCD) and fluorescence spectroscopy. In addition, some new developments, such as hetero-spectral 2DCOS, moving-window correlation, and model based correlation, are also reviewed for their utility in the investigation of the secondary structure, denaturation, folding and unfolding changes of protein. Finally, the new possibility and challenges of 2DCOS in protein research are highlighted as well.

Detection of rhodopsin dimerization in situ by PIE-FCCS, a time-resolved fluorescence spectroscopy.

PubMed

Smith, Adam W

2015-01-01

Rhodopsin self-associates in the plasma membrane. At low concentrations, the interactions are consistent with a monomer-dimer equilibrium (Comar et al., J Am Chem Soc 136(23):8342-8349, 2014). At high concentrations in native tissue, higher-order clusters have been observed (Fotiadis et al., Nature 421:127-128, 2003). The physiological role of rhodopsin dimerization is still being investigated, but it is clear that a quantitative assessment is essential to determining the function of rhodopsin clusters in vision. To quantify rhodopsin interactions, I will outline the theory and methodology of a specialized time-resolved fluorescence spectroscopy for measuring membrane protein-protein interactions called pulsed-interleaved excitation fluorescence cross-correlation spectroscopy (PIE-FCCS). The strength of this technique is its ability to quantify rhodopsin interactions in situ (i.e., a live cell plasma membrane). There are two reasons for restricting the scope to live cell membranes. First, the compositional heterogeneity of the plasma membrane creates a complex milieu with thousands of lipid, protein, and carbohydrate species. This makes it difficult to infer quaternary interactions from detergent solubilized samples or construct a model phospholipid bilayer that recapitulates all of the interactions present in native membranes. Second, organizational structure and dynamics is a key feature of the plasma membrane, and fixation techniques like formaldehyde cross-linking and vitrification will modulate the interactions. PIE-FCCS is based on two-color fluorescence imaging with time-correlated single-photon counting (TCSPC) (Becker et al., Rev Sci Instrum 70:1835-1841, 1999). By time-tagging every detected photon, the data can be analyzed as a fluorescence intensity distribution, fluorescence lifetime histogram, or fluorescence (cross-)correlation spectra (FCS/FCCS) (Becker, Advanced time-correlated single-photon counting techniques, Springer, Berlin, 2005). These analysis tools can then be used to quantify protein concentration, mobility, clustering, and Förster resonance energy transfer (FRET). In this paper I will focus on PIE-FCCS, which interleaves two wavelength excitation events in time so that the effects of spectral cross-talk and FRET can be isolated. In this way it is possible to characterize monomer-dimer-oligomer equilibria with high accuracy (Müller et al., Biophys J 89:3508-3522, 2005). Currently, PIE-FCCS requires a customized equipment configuration that will be described below. There is an excellent protocol that outlines traditional FCCS on a commercially available instrument (Bacia and Schwille, Nat Protoc 2:2842-2856, 2007). The PIE-FCCS approach is a relatively recent advance in FCCS that has been used in live cell assays to quantify lipid-anchored protein clustering (Triffo et al., J Am Chem Soc 134:10833-10842, 2012), epidermal growth factor receptor dimerization (Endres et al., Cell 152:543-556, 2013), and recently the dimerization of opsin (Comar et al., J Am Chem Soc 136(23):8342-8349, 2014). This paper will outline the theory and instrumentation requirements for PIE-FCCS, as well as the data collection and analysis process.

Three-color confocal Förster (or fluorescence) resonance energy transfer microscopy: Quantitative analysis of protein interactions in the nucleation of actin filaments in live cells.

PubMed

Wallrabe, Horst; Sun, Yuansheng; Fang, Xiaolan; Periasamy, Ammasi; Bloom, George S

2015-06-01

Experiments using live cell 3-color Förster (or fluorescence) resonance energy transfer (FRET) microscopy and corresponding in vitro biochemical reconstitution of the same proteins were conducted to evaluate actin filament nucleation. A novel application of 3-color FRET data is demonstrated, extending the analysis beyond the customary energy-transfer efficiency (E%) calculations. MDCK cells were transfected for coexpression of Teal-N-WASP/Venus-IQGAP1/mRFP1-Rac1, Teal-N-WASP/Venus-IQGAP1/mRFP1-Cdc42, CFP-Rac1/Venus-IQGAP1/mCherry-actin, or CFP-Cdc42/Venus-IQGAP1/mCherry-actin, and with single-label equivalents for spectral bleedthrough correction. Using confirmed E% as an entry point, fluorescence levels and related ratios were correlated at discrete accumulating levels at cell peripheries. Rising ratios of CFP-Rac1:Venus-IQGAP1 were correlated with lower overall actin fluorescence, whereas the CFP-Cdc42:Venus-IQGAP1 ratio correlated with increased actin fluorescence at low ratios, but was neutral at higher ratios. The new FRET analyses also indicated that rising levels of mRFP1-Cdc42 or mRFP1-Rac1, respectively, promoted or suppressed the association of Teal-N-WASP with Venus-IQGAP1. These 3-color FRET assays further support our in vitro results about the role of IQGAP1, Rac1, and Cdc42 in actin nucleation, and the differential impact of Rac1 and Cdc42 on the association of N-WASP with IQGAP1. In addition, this study emphasizes the power of 3-color FRET as a systems biology strategy for simultaneous evaluation of multiple interacting proteins in individual live cells. © 2015 International Society for Advancement of Cytometry.

Autofluorescence Lifetimes in Geographic Atrophy in Patients With Age-Related Macular Degeneration.

PubMed

Dysli, Chantal; Wolf, Sebastian; Zinkernagel, Martin S

2016-05-01

To investigate fluorescence lifetime characteristics in patients with geographic atrophy (GA) in eyes with age-related macular degeneration and to correlate the measurements with clinical data and optical coherence tomography (OCT) findings. Patients with GA were imaged with a fluorescence lifetime imaging ophthalmoscope. Retinal autofluorescence lifetimes were measured in a short and a long spectral channel (498-560 nm and 560-720 nm). Mean retinal fluorescence lifetimes were analyzed within GA and the surrounding retina, and data were correlated with best corrected visual acuity and OCT measurements. Fluorescence lifetime maps of 41 eyes of 41 patients (80 ± 7 years) with GA were analyzed. Mean lifetimes within areas of atrophy were prolonged by 624 ± 276 ps (+152%) in the short spectral channel and 418 ± 186 ps (+83%) in the long spectral channel compared to the surrounding tissue. Autofluorescence lifetime abnormalities in GA occurred with particular patterns, similar to those seen in fundus autofluorescence intensity images. Within the fovea short mean autofluorescence lifetimes were observed, presumably representing macular pigment. Short lifetimes were preserved even in the absence of foveal sparing but were decreased in patients with advanced retinal atrophy in OCT. Short lifetimes in the fovea correlated with better best corrected visual acuity in both spectral channels. This study established that autofluorescence lifetime changes in GA present with explicit patterns. We hypothesize that the short lifetimes seen within the atrophy may be used to estimate damage induced by atrophy and to monitor disease progression in the context of natural history or interventional therapeutic studies.

Visualization of Biosurfactant Film Flow in a Bacillus subtilis Swarm Colony on an Agar Plate

PubMed Central

Kim, Kyunghoon; Kim, Jung Kyung

2015-01-01

Collective bacterial dynamics plays a crucial role in colony development. Although many research groups have studied the behavior of fluidic swarm colonies, the detailed mechanics of its motion remains elusive. Here, we developed a visualization method using submicron fluorescent beads for investigating the flow field in a thin layer of fluid that covers a Bacillus subtilis swarm colony growing on an agar plate. The beads were initially embedded in the agar plate and subsequently distributed spontaneously at the upper surface of the expanding colony. We conducted long-term live cell imaging of the B. subtilis colony using the fluorescent tracers, and obtained high-resolution velocity maps of microscale vortices in the swarm colony using particle image velocimetry. A distinct periodic fluctuation in the average speed and vorticity of flow in swarm colony was observed at the inner region of the colony, and correlated with the switch between bacterial swarming and growth phases. At the advancing edge of the colony, both the magnitudes of velocity and vorticity of flow in swarm colony were inversely correlated with the spreading speed of the swarm edge. The advanced imaging tool developed in this study would facilitate further understanding of the effect of micro vortices in swarm colony on the collective dynamics of bacteria. PMID:26343634

Visualization of Biosurfactant Film Flow in a Bacillus subtilis Swarm Colony on an Agar Plate.

PubMed

Kim, Kyunghoon; Kim, Jung Kyung

2015-08-26

Collective bacterial dynamics plays a crucial role in colony development. Although many research groups have studied the behavior of fluidic swarm colonies, the detailed mechanics of its motion remains elusive. Here, we developed a visualization method using submicron fluorescent beads for investigating the flow field in a thin layer of fluid that covers a Bacillus subtilis swarm colony growing on an agar plate. The beads were initially embedded in the agar plate and subsequently distributed spontaneously at the upper surface of the expanding colony. We conducted long-term live cell imaging of the B. subtilis colony using the fluorescent tracers, and obtained high-resolution velocity maps of microscale vortices in the swarm colony using particle image velocimetry. A distinct periodic fluctuation in the average speed and vorticity of flow in swarm colony was observed at the inner region of the colony, and correlated with the switch between bacterial swarming and growth phases. At the advancing edge of the colony, both the magnitudes of velocity and vorticity of flow in swarm colony were inversely correlated with the spreading speed of the swarm edge. The advanced imaging tool developed in this study would facilitate further understanding of the effect of micro vortices in swarm colony on the collective dynamics of bacteria.

A retrospective analysis of compact fluorescent lamp experience curves and their correlations to deployment programs

DOE PAGES

Smith, Sarah Josephine; Wei, Max; Sohn, Michael D.

2016-09-17

Experience curves are useful for understanding technology development and can aid in the design and analysis of market transformation programs. Here, we employ a novel approach to create experience curves, to examine both global and North American compact fluorescent lamp (CFL) data for the years 1990–2007. We move away from the prevailing method of fitting a single, constant, exponential curve to data and instead search for break points where changes in the learning rate may have occurred. Our analysis suggests a learning rate of approximately 21% for the period of 1990–1997, and 51% and 79% in global and North Americanmore » datasets, respectively, after 1998. We use price data for this analysis; therefore our learning rates encompass developments beyond typical “learning by doing”, including supply chain impacts such as market competition. We examine correlations between North American learning rates and the initiation of new programs, abrupt technological advances, and economic and political events, and find an increased learning rate associated with design advancements and federal standards programs. Our findings support the use of segmented experience curves for retrospective and prospective technology analysis, and may imply that investments in technology programs have contributed to an increase of the CFL learning rate.« less

A retrospective analysis of compact fluorescent lamp experience curves and their correlations to deployment programs

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

Smith, Sarah Josephine; Wei, Max; Sohn, Michael D.

Experience curves are useful for understanding technology development and can aid in the design and analysis of market transformation programs. Here, we employ a novel approach to create experience curves, to examine both global and North American compact fluorescent lamp (CFL) data for the years 1990–2007. We move away from the prevailing method of fitting a single, constant, exponential curve to data and instead search for break points where changes in the learning rate may have occurred. Our analysis suggests a learning rate of approximately 21% for the period of 1990–1997, and 51% and 79% in global and North Americanmore » datasets, respectively, after 1998. We use price data for this analysis; therefore our learning rates encompass developments beyond typical “learning by doing”, including supply chain impacts such as market competition. We examine correlations between North American learning rates and the initiation of new programs, abrupt technological advances, and economic and political events, and find an increased learning rate associated with design advancements and federal standards programs. Our findings support the use of segmented experience curves for retrospective and prospective technology analysis, and may imply that investments in technology programs have contributed to an increase of the CFL learning rate.« less

Robust Bayesian Fluorescence Lifetime Estimation, Decay Model Selection and Instrument Response Determination for Low-Intensity FLIM Imaging

PubMed Central

Rowley, Mark I.; Coolen, Anthonius C. C.; Vojnovic, Borivoj; Barber, Paul R.

2016-01-01

We present novel Bayesian methods for the analysis of exponential decay data that exploit the evidence carried by every detected decay event and enables robust extension to advanced processing. Our algorithms are presented in the context of fluorescence lifetime imaging microscopy (FLIM) and particular attention has been paid to model the time-domain system (based on time-correlated single photon counting) with unprecedented accuracy. We present estimates of decay parameters for mono- and bi-exponential systems, offering up to a factor of two improvement in accuracy compared to previous popular techniques. Results of the analysis of synthetic and experimental data are presented, and areas where the superior precision of our techniques can be exploited in Förster Resonance Energy Transfer (FRET) experiments are described. Furthermore, we demonstrate two advanced processing methods: decay model selection to choose between differing models such as mono- and bi-exponential, and the simultaneous estimation of instrument and decay parameters. PMID:27355322

Determination of HER2 amplification in primary breast cancer using dual-colour chromogenic in situ hybridization is comparable to fluorescence in situ hybridization: a European multicentre study involving 168 specimens.

PubMed

García-Caballero, Tomás; Grabau, Dorthe; Green, Andrew R; Gregory, John; Schad, Arno; Kohlwes, Elke; Ellis, Ian O; Watts, Sarah; Mollerup, Jens

2010-03-01

Fluorescence in situ hybridization (FISH) can be used to reveal several genomic imbalances relevant to proper cancer diagnosis and to the correct treatment regime. However, FISH requires expensive and advanced fluorescence microscopes in addition to expertise in fluorescence microscopy. To determine whether a newly developed dual-colour chromogenic in situ hybridization (CISH) method is a suitable alternative to FISH, we analysed the human epidermal growth factor receptor 2 gene (HER2) amplification level of 168 breast cancer specimens using dual-colour CISH and FISH and compared the results. We found 100% agreement between HER2 status determined by FISH and dual-colour CISH. Furthermore, we observed that the time used to score slides was significantly reduced by 28% in dual-colour CISH compared with the FISH protocol. Concordance between HER2 protein status and dual-colour CISH or FISH was equally good with an overall agreement of 96.8%. Correlation between the HER2/centromere 17 gene ratios obtained with dual-colour CISH and FISH was highly significant with an overall correlation coefficient (rho) of 0.96. We conclude that dual-colour CISH and bright field microscopy are excellent alternatives to FISH when analysing the HER2 status of primary breast cancer.

Recent advancement in the field of two-dimensional correlation spectroscopy

NASA Astrophysics Data System (ADS)

Noda, Isao

2008-07-01

The recent advancement in the field of 2D correlation spectroscopy is reviewed with the emphasis on a number of papers published during the last two years. Topics covered by this comprehensive review include books, review articles, and noteworthy developments in the theory and applications of 2D correlation spectroscopy. New 2D correlation techniques are discussed, such as kernel analysis and augmented 2D correlation, model-based correlation, moving window analysis, global phase angle, covariance and correlation coefficient mapping, sample-sample correlation, hybrid and hetero correlation, pretreatment and transformation of data, and 2D correlation combined with other chemometrics techniques. Perturbation methods of both static (e.g., temperature, composition, pressure and stress, spatial distribution and orientation) and dynamic types (e.g., rheo-optical and acoustic, chemical reactions and kinetics, H/D exchange, sorption and diffusion) currently in use are examined. Analytical techniques most commonly employed in 2D correlation spectroscopy are IR, Raman, and NIR, but the growing use of other probes is also noted, including fluorescence, emission, Raman optical activity and vibrational circular dichroism, X-ray absorption and scattering, NMR, mass spectrometry, and even chromatography. The field of applications for 2D correlation spectroscopy is very diverse, encompassing synthetic polymers, liquid crystals, Langmuir-Blodgett films, proteins and peptides, natural polymers and biomaterials, pharmaceuticals, food and agricultural products, water, solutions, inorganic, organic, hybrid or composite materials, and many more.

Recent Advances in Silicon Nanomaterial-Based Fluorescent Sensors.

PubMed

Wang, Houyu; He, Yao

2017-02-03

During the past decades, owing to silicon nanomaterials' unique optical properties, benign biocompatibility, and abundant surface chemistry, different dimensional silicon nanostructures have been widely employed for rationally designing and fabricating high-performance fluorescent sensors for the detection of various chemical and biological species. Among of these, zero-dimensional silicon nanoparticles (SiNPs) and one-dimensional silicon nanowires (SiNWs) are of particular interest. Herein, we focus on reviewing recent advances in silicon nanomaterials-based fluorescent sensors from a broad perspective and discuss possible future directions. Firstly, we introduce the latest achievement of zero-dimensional SiNP-based fluorescent sensors. Next, we present recent advances of one-dimensional SiNW-based fluorescent sensors. Finally, we discuss the major challenges and prospects for the development of silicon-based fluorescent sensors.

Recent Advances in Silicon Nanomaterial-Based Fluorescent Sensors

PubMed Central

Wang, Houyu; He, Yao

2017-01-01

During the past decades, owing to silicon nanomaterials’ unique optical properties, benign biocompatibility, and abundant surface chemistry, different dimensional silicon nanostructures have been widely employed for rationally designing and fabricating high-performance fluorescent sensors for the detection of various chemical and biological species. Among of these, zero-dimensional silicon nanoparticles (SiNPs) and one-dimensional silicon nanowires (SiNWs) are of particular interest. Herein, we focus on reviewing recent advances in silicon nanomaterials-based fluorescent sensors from a broad perspective and discuss possible future directions. Firstly, we introduce the latest achievement of zero-dimensional SiNP-based fluorescent sensors. Next, we present recent advances of one-dimensional SiNW-based fluorescent sensors. Finally, we discuss the major challenges and prospects for the development of silicon-based fluorescent sensors. PMID:28165357

ultraLM and miniLM: Locator tools for smart tracking of fluorescent cells in correlative light and electron microscopy.

PubMed

Brama, Elisabeth; Peddie, Christopher J; Wilkes, Gary; Gu, Yan; Collinson, Lucy M; Jones, Martin L

2016-12-13

In-resin fluorescence (IRF) protocols preserve fluorescent proteins in resin-embedded cells and tissues for correlative light and electron microscopy, aiding interpretation of macromolecular function within the complex cellular landscape. Dual-contrast IRF samples can be imaged in separate fluorescence and electron microscopes, or in dual-modality integrated microscopes for high resolution correlation of fluorophore to organelle. IRF samples also offer a unique opportunity to automate correlative imaging workflows. Here we present two new locator tools for finding and following fluorescent cells in IRF blocks, enabling future automation of correlative imaging. The ultraLM is a fluorescence microscope that integrates with an ultramicrotome, which enables 'smart collection' of ultrathin sections containing fluorescent cells or tissues for subsequent transmission electron microscopy or array tomography. The miniLM is a fluorescence microscope that integrates with serial block face scanning electron microscopes, which enables 'smart tracking' of fluorescent structures during automated serial electron image acquisition from large cell and tissue volumes.

Time-Domain Microfluidic Fluorescence Lifetime Flow Cytometry for High-Throughput Förster Resonance Energy Transfer Screening

PubMed Central

Nedbal, Jakub; Visitkul, Viput; Ortiz-Zapater, Elena; Weitsman, Gregory; Chana, Prabhjoat; Matthews, Daniel R; Ng, Tony; Ameer-Beg, Simon M

2015-01-01

Sensing ion or ligand concentrations, physico-chemical conditions, and molecular dimerization or conformation change is possible by assays involving fluorescent lifetime imaging. The inherent low throughput of imaging impedes rigorous statistical data analysis on large cell numbers. We address this limitation by developing a fluorescence lifetime-measuring flow cytometer for fast fluorescence lifetime quantification in living or fixed cell populations. The instrument combines a time-correlated single photon counting epifluorescent microscope with microfluidics cell-handling system. The associated computer software performs burst integrated fluorescence lifetime analysis to assign fluorescence lifetime, intensity, and burst duration to each passing cell. The maximum safe throughput of the instrument reaches 3,000 particles per minute. Living cells expressing spectroscopic rulers of varying peptide lengths were distinguishable by Förster resonant energy transfer measured by donor fluorescence lifetime. An epidermal growth factor (EGF)-stimulation assay demonstrated the technique's capacity to selectively quantify EGF receptor phosphorylation in cells, which was impossible by measuring sensitized emission on a standard flow cytometer. Dual-color fluorescence lifetime detection and cell-specific chemical environment sensing were exemplified using di-4-ANEPPDHQ, a lipophilic environmentally sensitive dye that exhibits changes in its fluorescence lifetime as a function of membrane lipid order. To our knowledge, this instrument opens new applications in flow cytometry which were unavailable due to technological limitations of previously reported fluorescent lifetime flow cytometers. The presented technique is sensitive to lifetimes of most popular fluorophores in the 0.5–5 ns range including fluorescent proteins and is capable of detecting multi-exponential fluorescence lifetime decays. This instrument vastly enhances the throughput of experiments involving fluorescence lifetime measurements, thereby providing statistically significant quantitative data for analysis of large cell populations. © 2014 International Society for Advancement of Cytometry PMID:25523156

Low-cost fluorescence microscopy for point-of-care cell imaging

NASA Astrophysics Data System (ADS)

Lochhead, Michael J.; Ives, Jeff; Givens, Monique; Delaney, Marie; Moll, Kevin; Myatt, Christopher J.

2010-02-01

Fluorescence microscopy has long been a standard tool in laboratory medicine. Implementation of fluorescence microscopy for near-patient diagnostics, however, has been limited due to cost and complexity associated with traditional fluorescence microscopy techniques. There is a particular need for robust, low-cost imaging in high disease burden areas in the developing world, where access to central laboratory facilities and trained staff is limited. Here we describe a point-of-care assay that combines a disposable plastic cartridge with an extremely low cost fluorescence imaging instrument. Based on a novel, multi-mode planar waveguide configuration, the system capitalizes on advances in volume-manufactured consumer electronic components to deliver an imaging system with minimal moving parts and low power requirements. A two-color cell imager is presented, with magnification optimized for enumeration of immunostained human T cells. To demonstrate the system, peripheral blood mononuclear cells were stained with fluorescently labeled anti-human-CD4 and anti-human-CD3 antibodies. Registered images were used to generate fractional CD4+ and CD3+ staining and enumeration results that show excellent correlation with flow cytometry. The cell imager is under development as a very low cost CD4+ T cell counter for HIV disease management in limited resource settings.

Novel fluorescence molecular imaging of chemotherapy-induced intestinal apoptosis

NASA Astrophysics Data System (ADS)

Levin, Galit; Shirvan, Anat; Grimberg, Hagit; Reshef, Ayelet; Yogev-Falach, Merav; Cohen, Avi; Ziv, Ilan

2009-09-01

Chemotherapy-induced enteropathy (CIE) is one of the most serious complications of anticancer therapy, and tools for its early detection and monitoring are highly needed. We report on a novel fluorescence method for detection of CIE, based on molecular imaging of the related apoptotic process. The method comprises systemic intravenous administration of the ApoSense fluorescent biomarker (N,N'-didansyl-L-cystine DDC) in vivo and subsequent fluorescence imaging of the intestinal mucosa. In the reported proof-of-concept studies, mice were treated with either taxol+cyclophosphamide or doxil. DDC was administered in vivo at various time points after drug administration, and tracer uptake by ileum tissue was subsequently evaluated by ex vivo fluorescent microscopy. Chemotherapy caused marked and selective uptake of DDC in ileal epithelial cells, in correlation with other hallmarks of apoptosis (i.e., DNA fragmentation and Annexin-V binding). Induction of DDC uptake occurred early after chemotherapy, and its temporal profile was parallel to that of the apoptotic process, as assessed histologically. DDC may therefore serve as a useful tool for detection of CIE. Future potential integration of this method with fluorescent endoscopic techniques, or development of radio-labeled derivatives of DDC for emission tomography, may advance early diagnosis and monitoring of this severe adverse effect of chemotherapy.

Methylglyoxal induced glycation and aggregation of human serum albumin: Biochemical and biophysical approach.

PubMed

Ahmed, Azaj; Shamsi, Anas; Khan, Mohd Shahnawaz; Husain, Fohad Mabood; Bano, Bilqees

2018-07-01

Serum protein glycation and formation of advanced glycation end products (AGEs) correlates with many diseases viz. diabetes signifying the importance of studying the glycation pattern of serum proteins. In our present study, methylglyoxal was investigated for its effect on the structure of human serum albumin (HSA); exploring the formation of AGEs and aggregates of HSA. The analytical tools employed includes intrinsic and extrinsic fluorescence, UV spectroscopy, far UV circular dichroism, Thioflavin T fluorescence, congo red binding, polyacrylamide gel electrophoresis (PAGE). UV and fluorescence spectroscopy revealed the structural transition of native HSA evident by new peaks and increased absorbance in UV spectra and quenched fluorescence in the presence of MG. Far UV CD spectroscopy revealed MG induced secondary structural alteration evident by reduced α-helical content. AGEs formation was confirmed by AGEs specific fluorescence. Increased ThT fluorescence and CR absorbance of 10mM MG incubated HSA suggests that glycated HSA results in the formation of aggregates of HSA. SEM and TEM were reported to have an insight of these aggregates. Molecular docking was also utilized to see site specific interaction of MG-HSA. This study is clinically significant as HSA is a clinically relevant protein which plays a crucial role in many diseases. Copyright © 2018 Elsevier B.V. All rights reserved.

Formation of immunochemical advanced glycosylation end products precedes and correlates with early manifestations of renal and retinal disease in diabetes.

PubMed

Beisswenger, P J; Makita, Z; Curphey, T J; Moore, L L; Jean, S; Brinck-Johnsen, T; Bucala, R; Vlassara, H

1995-07-01

Elevated levels of advanced glycosylation end products (AGEs) have been found in multiple tissues in association with diabetic vascular complications and during the microalbuminuric phase of diabetic nephropathy. In this study, we have used an AGE-specific enzyme-linked immunosorbent assay (ELISA) to measure skin AGEs to determine whether elevated levels can be detected before the onset of overt microangiopathy. Subjects with type I diabetes (n = 48) were graded for the degree of nephropathy (normal [23], microalbuminuria [12], or macroalbuminuria [12]) and retinopathy (none [13], background [20], or proliferative [15]). Subgroups with a premicroalbuminuric phase of albumin excretion (< or = 28 mg/24 h, n = 27) or with the earliest stages of retinopathy (n = 27) were identified. A significant increase in tissue AGEs was found as urinary albumin increased during the premicroalbuminuric phase of nephropathy even when the data were adjusted for age and duration of diabetes (P = 0.005). Immunoreactive AGEs also increased as normal renal status advanced to microalbuminuria and macroalbuminuria (P = 0.0001 across groups). Significant elevation of AGEs was also found in association with the earliest stages of clinically evident retinopathy (early background versus minimal grades). In addition, higher AGE levels were found in subjects with proliferative retinopathy when compared with those with less severe retinopathy (P < 0.004 across groups). In contrast, no significant differences were found in tissue AGE levels between groups with or without early retinopathy based on pentosidine or fluorescent AGE measurements, although fluorescent AGEs correlated with albumin excretion.(ABSTRACT TRUNCATED AT 250 WORDS)

A Multi-Functional Imaging Approach to High-Content Protein Interaction Screening

PubMed Central

Matthews, Daniel R.; Fruhwirth, Gilbert O.; Weitsman, Gregory; Carlin, Leo M.; Ofo, Enyinnaya; Keppler, Melanie; Barber, Paul R.; Tullis, Iain D. C.; Vojnovic, Borivoj; Ng, Tony; Ameer-Beg, Simon M.

2012-01-01

Functional imaging can provide a level of quantification that is not possible in what might be termed traditional high-content screening. This is due to the fact that the current state-of-the-art high-content screening systems take the approach of scaling-up single cell assays, and are therefore based on essentially pictorial measures as assay indicators. Such phenotypic analyses have become extremely sophisticated, advancing screening enormously, but this approach can still be somewhat subjective. We describe the development, and validation, of a prototype high-content screening platform that combines steady-state fluorescence anisotropy imaging with fluorescence lifetime imaging (FLIM). This functional approach allows objective, quantitative screening of small molecule libraries in protein-protein interaction assays. We discuss the development of the instrumentation, the process by which information on fluorescence resonance energy transfer (FRET) can be extracted from wide-field, acceptor fluorescence anisotropy imaging and cross-checking of this modality using lifetime imaging by time-correlated single-photon counting. Imaging of cells expressing protein constructs where eGFP and mRFP1 are linked with amino-acid chains of various lengths (7, 19 and 32 amino acids) shows the two methodologies to be highly correlated. We validate our approach using a small-scale inhibitor screen of a Cdc42 FRET biosensor probe expressed in epidermoid cancer cells (A431) in a 96 microwell-plate format. We also show that acceptor fluorescence anisotropy can be used to measure variations in hetero-FRET in protein-protein interactions. We demonstrate this using a screen of inhibitors of internalization of the transmembrane receptor, CXCR4. These assays enable us to demonstrate all the capabilities of the instrument, image processing and analytical techniques that have been developed. Direct correlation between acceptor anisotropy and donor FLIM is observed for FRET assays, providing an opportunity to rapidly screen proteins, interacting on the nano-meter scale, using wide-field imaging. PMID:22506000

Hydrodynamic boundary condition of water on hydrophobic surfaces.

PubMed

Schaeffel, David; Yordanov, Stoyan; Schmelzeisen, Marcus; Yamamoto, Tetsuya; Kappl, Michael; Schmitz, Roman; Dünweg, Burkhard; Butt, Hans-Jürgen; Koynov, Kaloian

2013-05-01

By combining total internal reflection fluorescence cross-correlation spectroscopy with Brownian dynamics simulations, we were able to measure the hydrodynamic boundary condition of water flowing over a smooth solid surface with exceptional accuracy. We analyzed the flow of aqueous electrolytes over glass coated with a layer of poly(dimethylsiloxane) (advancing contact angle Θ = 108°) or perfluorosilane (Θ = 113°). Within an error of better than 10 nm the slip length was indistinguishable from zero on all surfaces.

Intracellular applications of fluorescence correlation spectroscopy: prospects for neuroscience.

PubMed

Kim, Sally A; Schwille, Petra

2003-10-01

Based on time-averaging fluctuation analysis of small fluorescent molecular ensembles in equilibrium, fluorescence correlation spectroscopy has recently been applied to investigate processes in the intracellular milieu. The exquisite sensitivity of fluorescence correlation spectroscopy provides access to a multitude of measurement parameters (rates of diffusion, local concentration, states of aggregation and molecular interactions) in real time with fast temporal and high spatial resolution. The introduction of dual-color cross-correlation, imaging, two-photon excitation, and coincidence analysis coupled with fluorescence correlation spectroscopy has expanded the utility of the technique to encompass a wide range of promising applications in living cells that may provide unprecedented insight into understanding the molecular mechanisms of intracellular neurobiological processes.

Molecular Diffusion in Plasma Membranes of Primary Lymphocytes Measured by Fluorescence Correlation Spectroscopy.

PubMed

Staaf, Elina; Bagawath-Singh, Sunitha; Johansson, Sofia

2017-02-01

Fluorescence correlation spectroscopy (FCS) is a powerful technique for studying the diffusion of molecules within biological membranes with high spatial and temporal resolution. FCS can quantify the molecular concentration and diffusion coefficient of fluorescently labeled molecules in the cell membrane. This technique has the ability to explore the molecular diffusion characteristics of molecules in the plasma membrane of immune cells in steady state (i.e., without processes affecting the result during the actual measurement time). FCS is suitable for studying the diffusion of proteins that are expressed at levels typical for most endogenous proteins. Here, a straightforward and robust method to determine the diffusion rate of cell membrane proteins on primary lymphocytes is demonstrated. An effective way to perform measurements on antibody-stained live cells and commonly occurring observations after acquisition are described. The recent advancements in the development of photo-stable fluorescent dyes can be utilized by conjugating the antibodies of interest to appropriate dyes that do not bleach extensively during the measurements. Additionally, this allows for the detection of slowly diffusing entities, which is a common feature of proteins expressed in cell membranes. The analysis procedure to extract molecular concentration and diffusion parameters from the generated autocorrelation curves is highlighted. In summary, a basic protocol for FCS measurements is provided; it can be followed by immunologists with an understanding of confocal microscopy but with no other previous experience of techniques for measuring dynamic parameters, such as molecular diffusion rates.

Using Multiorder Time-Correlation Functions (TCFs) To Elucidate Biomolecular Reaction Pathways from Microsecond Single-Molecule Fluorescence Experiments.

PubMed

Phelps, Carey; Israels, Brett; Marsh, Morgan C; von Hippel, Peter H; Marcus, Andrew H

2016-12-29

Recent advances in single-molecule fluorescence imaging have made it possible to perform measurements on microsecond time scales. Such experiments have the potential to reveal detailed information about the conformational changes in biological macromolecules, including the reaction pathways and dynamics of the rearrangements involved in processes, such as sequence-specific DNA "breathing" and the assembly of protein-nucleic acid complexes. Because microsecond-resolved single-molecule trajectories often involve "sparse" data, that is, they contain relatively few data points per unit time, they cannot be easily analyzed using the standard protocols that were developed for single-molecule experiments carried out with tens-of-millisecond time resolution and high "data density." Here, we describe a generalized approach, based on time-correlation functions, to obtain kinetic information from microsecond-resolved single-molecule fluorescence measurements. This approach can be used to identify short-lived intermediates that lie on reaction pathways connecting relatively long-lived reactant and product states. As a concrete illustration of the potential of this methodology for analyzing specific macromolecular systems, we accompany the theoretical presentation with the description of a specific biologically relevant example drawn from studies of reaction mechanisms of the assembly of the single-stranded DNA binding protein of the T4 bacteriophage replication complex onto a model DNA replication fork.

Optimization of advanced Wiener estimation methods for Raman reconstruction from narrow-band measurements in the presence of fluorescence background

PubMed Central

Chen, Shuo; Ong, Yi Hong; Lin, Xiaoqian; Liu, Quan

2015-01-01

Raman spectroscopy has shown great potential in biomedical applications. However, intrinsically weak Raman signals cause slow data acquisition especially in Raman imaging. This problem can be overcome by narrow-band Raman imaging followed by spectral reconstruction. Our previous study has shown that Raman spectra free of fluorescence background can be reconstructed from narrow-band Raman measurements using traditional Wiener estimation. However, fluorescence-free Raman spectra are only available from those sophisticated Raman setups capable of fluorescence suppression. The reconstruction of Raman spectra with fluorescence background from narrow-band measurements is much more challenging due to the significant variation in fluorescence background. In this study, two advanced Wiener estimation methods, i.e. modified Wiener estimation and sequential weighted Wiener estimation, were optimized to achieve this goal. Both spontaneous Raman spectra and surface enhanced Raman spectra were evaluated. Compared with traditional Wiener estimation, two advanced methods showed significant improvement in the reconstruction of spontaneous Raman spectra. However, traditional Wiener estimation can work as effectively as the advanced methods for SERS spectra but much faster. The wise selection of these methods would enable accurate Raman reconstruction in a simple Raman setup without the function of fluorescence suppression for fast Raman imaging. PMID:26203387

Reconstructed Solar-Induced Fluorescence: A Machine Learning Vegetation Product Based on MODIS Surface Reflectance to Reproduce GOME-2 Solar-Induced Fluorescence

NASA Astrophysics Data System (ADS)

Gentine, P.; Alemohammad, S. H.

2018-04-01

Solar-induced fluorescence (SIF) observations from space have resulted in major advancements in estimating gross primary productivity (GPP). However, current SIF observations remain spatially coarse, infrequent, and noisy. Here we develop a machine learning approach using surface reflectances from Moderate Resolution Imaging Spectroradiometer (MODIS) channels to reproduce SIF normalized by clear sky surface irradiance from the Global Ozone Monitoring Experiment-2 (GOME-2). The resulting product is a proxy for ecosystem photosynthetically active radiation absorbed by chlorophyll (fAPARCh). Multiplying this new product with a MODIS estimate of photosynthetically active radiation provides a new MODIS-only reconstruction of SIF called Reconstructed SIF (RSIF). RSIF exhibits much higher seasonal and interannual correlation than the original SIF when compared with eddy covariance estimates of GPP and two reference global GPP products, especially in dry and cold regions. RSIF also reproduces intense productivity regions such as the U.S. Corn Belt contrary to typical vegetation indices and similarly to SIF.

The bright future of single-molecule fluorescence imaging

PubMed Central

Juette, Manuel F.; Terry, Daniel S.; Wasserman, Michael R.; Zhou, Zhou; Altman, Roger B.; Zheng, Qinsi; Blanchard, Scott C.

2014-01-01

Single-molecule Förster resonance energy transfer (smFRET) is an essential and maturing tool to probe biomolecular interactions and conformational dynamics in vitro and, increasingly, in living cells. Multi-color smFRET enables the correlation of multiple such events and the precise dissection of their order and timing. However, the requirements for good spectral separation, high time resolution, and extended observation times place extraordinary demands on the fluorescent labels used in such experiments. Together with advanced experimental designs and data analysis, the development of long-lasting, non-fluctuating fluorophores is therefore proving key to progress in the field. Recently developed strategies for obtaining ultra-stable organic fluorophores spanning the visible spectrum are underway that will enable multi-color smFRET studies to deliver on their promise of previously unachievable biological insights. PMID:24956235

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.

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;

Correlative nanoscale imaging of actin filaments and their complexes

NASA Astrophysics Data System (ADS)

Sharma, Shivani; Zhu, Huanqi; Grintsevich, Elena E.; Reisler, Emil; Gimzewski, James K.

2013-06-01

Actin remodeling is an area of interest in biology in which correlative microscopy can bring a new way to analyze protein complexes at the nanoscale. Advances in EM, X-ray diffraction, fluorescence, and single molecule techniques have provided a wealth of information about the modulation of the F-actin structure and its regulation by actin binding proteins (ABPs). Yet, there are technological limitations of these approaches to achieving quantitative molecular level information on the structural and biophysical changes resulting from ABPs interaction with F-actin. Fundamental questions about the actin structure and dynamics and how these determine the function of ABPs remain unanswered. Specifically, how local and long-range structural and conformational changes result in ABPs induced remodeling of F-actin needs to be addressed at the single filament level. Advanced, sensitive and accurate experimental tools for detailed understanding of ABP-actin interactions are much needed. This article discusses the current understanding of nanoscale structural and mechanical modulation of F-actin by ABPs at the single filament level using several correlative microscopic techniques, focusing mainly on results obtained by Atomic Force Microscopy (AFM) analysis of ABP-actin complexes.

Measuring Photosynthetic Response to Drought Stress using Active and Passive Fluorescence

NASA Astrophysics Data System (ADS)

Helm, L.; Lerdau, M.; Wang, W.; Yang, X.

2017-12-01

Photosynthesis, the endothermic reactions involving the absorption of light and fixation and reduction of carbon dioxide by plants, plays important roles in carbon and water cycles, food security, and even weather and climate patterns. Solar radiation provides the energy for photosynthesis, but often plants absorb more solar energy than they can use to reduce carbon dioxide. This excess energy, which is briefly stored as high-energy electrons in the chloroplast, must be removed or damage to the leaf's photosynthetic machinery will occur. One important energy dissipation pathway is for the high energy electrons to return to their lower valance state and, in doing so, release radiation (fluorescence). This fluorescence (known as solar induced fluorescence (SIF) has been found to strongly correlate with gross photosynthesis. Recent advances in the remote sensing of SIF allow for large-scale real-time estimation of photosynthesis. In a warming climate with more frequent stress, remote sensing is necessary for measuring the spatial and temporal variability of photosynthesis. However, the mechanisms that link SIF and photosynthesis are unclear, particularly how the relationship may or may not change under stress. We present data from leaf-level measurements of gas exchange, pulse amplitude modulation (PAM) fluorescence, and SIF in two major tree species in North America. Water-stressed and well-watered plants were compared to determine how SIF and carbon dioxide exchange are modulated by drought diurnally and seasonally. Secondly, photosynthesis and fluorescence under high and low oxygen concentrations were compared to determine how photorespiration alters the relationship between SIF and gross photosynthesis. We find a strong correlation between SIF and steady-state fluorescence measured with conventional PAM fluorometry. Our results also indicate that drought-stress modulates the SIF-photosynthesis relationship, and this may be driven by drought-induced changes in stomatal conductance that change the relationship between photosynthesis and photorespiration. We also show a response to drought stress measured with active and passive fluorescence. Application of these findings will allow remote sensing of SIF to be utilized on a larger scale.

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.

In vivo fluorescence lifetime imaging for monitoring the efficacy of the cancer treatment.

PubMed

Ardeshirpour, Yasaman; Chernomordik, Victor; Hassan, Moinuddin; Zielinski, Rafal; Capala, Jacek; Gandjbakhche, Amir

2014-07-01

Advances in tumor biology created a foundation for targeted therapy aimed at inactivation of specific molecular mechanisms responsible for cell malignancy. In this paper, we used in vivo fluorescence lifetime imaging with HER2-targeted fluorescent probes as an alternative imaging method to investigate the efficacy of targeted therapy with 17-DMAG (an HSP90 inhibitor) on tumors with high expression of HER2 receptors. HER2-specific Affibody, conjugated to Alexafluor 750, was injected into nude mice bearing HER2-positive tumor xenograft. The fluorescence lifetime was measured before treatment and monitored after the probe injections at 12 hours after the last treatment dose, when the response to the 17-DMAG therapy was the most pronounced as well as a week after the last treatment when the tumors grew back almost to their pretreatment size. Imaging results showed significant difference between the fluorescence lifetimes at the tumor and the contralateral site (∼0.13 ns) in the control group (before treatment) and 7 days after the last treatment when the tumors grew back to their pretreatment dimensions. However, at the time frame that the treatment had its maximum effect (12 hours after the last treatment), the difference between the fluorescence lifetime at the tumor and contralateral site decreased to 0.03 ns. The results showed a good correlation between fluorescence lifetime and the efficacy of the treatment. These findings show that in vivo fluorescence lifetime imaging can be used as a promising molecular imaging tool for monitoring the treatment outcome in preclinical models and potentially in patients. ©2014 American Association for Cancer Research.

In-vivo fluorescence lifetime imaging for monitoring the efficacy of the cancer treatment

PubMed Central

Ardeshirpour, Yasaman; Chernomordik, Victor; Hassan, Moinuddin; Zielinski, Rafal; Capala, Jacek; Gandjbakhche, Amir

2015-01-01

Purpose Advances in tumor biology created a foundation for targeted therapy aimed at inactivation of specific molecular mechanisms responsible for cell malignancy. In this paper, we used in-vivo fluorescence lifetime imaging with HER2 targeted fluorescent probes as an alternative imaging method to investigate the efficacy of targeted therapy with 17-DMAG (an HSP90 inhibitor) on tumors with high expression of HER2 receptors. Experimental Design HER2-specific Affibody, conjugated to Alexafluor 750, was injected into nude mice, bearing HER2-positive tumor xenograft. The fluorescence lifetime was measured before treatment and monitored after the probe injections at 12 hours after the last treatment dose, when the response to the 17-DMAG therapy was the most pronounced as well as a week after the last treatment when the tumors grew back almost to their pre-treatment size. Results Imaging results showed significant difference between the fluorescence lifetimes at the tumor and the contralateral site (~0.13ns) in the control group (before treatment) and 7 days after the last treatment when the tumors grew back to their pretreatment dimensions. However, at the time frame that the treatment had its maximum effect (12 hours after the last treatment) the difference between the fluorescence lifetime at the tumor and contralateral site decreased to 0.03ns. Conclusions The results showed a good correlation between fluorescence lifetime and the efficacy of the treatment. These findings show that in-vivo fluorescence lifetime imaging can be used as a promising molecular imaging tool for monitoring the treatment outcome in preclinical models and potentially in patients. PMID:24671949

Deep brain optical measurements of cell type-specific neural activity in behaving mice.

PubMed

Cui, Guohong; Jun, Sang Beom; Jin, Xin; Luo, Guoxiang; Pham, Michael D; Lovinger, David M; Vogel, Steven S; Costa, Rui M

2014-01-01

Recent advances in genetically encoded fluorescent sensors enable the monitoring of cellular events from genetically defined groups of neurons in vivo. In this protocol, we describe how to use a time-correlated single-photon counting (TCSPC)-based fiber optics system to measure the intensity, emission spectra and lifetime of fluorescent biosensors expressed in deep brain structures in freely moving mice. When combined with Cre-dependent selective expression of genetically encoded Ca(2+) indicators (GECIs), this system can be used to measure the average neural activity from a specific population of cells in mice performing complex behavioral tasks. As an example, we used viral expression of GCaMPs in striatal projection neurons (SPNs) and recorded the fluorescence changes associated with calcium spikes from mice performing a lever-pressing operant task. The whole procedure, consisting of virus injection, behavior training and optical recording, takes 3-4 weeks to complete. With minor adaptations, this protocol can also be applied to recording cellular events from other cell types in deep brain regions, such as dopaminergic neurons in the ventral tegmental area. The simultaneously recorded fluorescence signals and behavior events can be used to explore the relationship between the neural activity of specific brain circuits and behavior.

Characterizing chlorine oxidation of dissolved organic matter and disinfection by-product formation with fluorescence spectroscopy and parallel factor analysis

NASA Astrophysics Data System (ADS)

Beggs, Katherine M. H.; Summers, R. Scott; McKnight, Diane M.

2009-12-01

Relationships between chlorine demand and disinfection by-product (DBP) formation during chlorination and fluorescence of dissolved organic matter (DOM) were developed. Fluorescence excitation and emission (EEM) spectroscopy was employed, and parameters including fluorescence index, redox index, and overall fluorescence intensity (OFI) were correlated to chlorine demand and DBP formation. The EEMs were also analyzed using a well established global parallel factor analysis (PARAFAC) model which resolves the fluorescence signal into 13 components, including quinone-like and protein-like components. Over an 8-day chlorination period the OFI and sum of the 13 PARAFAC loadings decreased by more than 70%. The remaining identified quinone-like compounds within the DOM were shifted to a more oxidized state. Quinone fluorescence was strongly correlated to both reduced fluorescence intensity and to chlorine demand which indicates that fluorescence may be used to track the chlorine oxidation of DOM. Quinone fluorescence was also correlated strongly with both classes of regulated DBPs: total trihalomethanes and haloacetic acids. Quinone-like components were found to be strongly correlated to overall, short-term, and long-term specific DBP formation. The results of this study show that fluorescence is a useful tool in tracking both DOM oxidation and DBP formation during chlorination.

Hard X-ray Fluorescence Microscopy to Determine the Element Distribution of Soil Colloids in Aqueous Environment

NASA Astrophysics Data System (ADS)

Gleber, S.-C.; Vogt, S.; Niemeyer, J.; Finney, L.; McNulty, I.; Thieme, J.

2011-09-01

A prominent feature of soil colloids is their huge specific surface. It determines colloidal properties such as adsorption capacity or diffusion. The colloidal interactions differ significantly from the behavior of the same materials in a bulk system. Interactions in the colloidal regime are crucial, for example, for the transport and release of nutrients and toxicants in soils, which then influences directly the growth of plants. However, there is still a need for more analytical resources to study those interactions. To reveal the correlation of the particular trace elements and their distribution in correlation to colloidal interactions as well as changing pH values, experiments at the hard x-ray fluorescence microprobe at beamline 2-ID-E of the Advanced Photon Source (APS), were performed with colloidal clay and soil samples in an aqueous environment as naturally relevant. To obtain further spatial information, stereo imaging has been used. To study the dynamical behavior of these colloidal suspensions at changing pH, a wet sample chamber allowing in situ manipulation was developed and utilized.

Maximum entropy analysis of polarized fluorescence decay of (E)GFP in aqueous solution

NASA Astrophysics Data System (ADS)

Novikov, Eugene G.; Skakun, Victor V.; Borst, Jan Willem; Visser, Antonie J. W. G.

2018-01-01

The maximum entropy method (MEM) was used for the analysis of polarized fluorescence decays of enhanced green fluorescent protein (EGFP) in buffered water/glycerol mixtures, obtained with time-correlated single-photon counting (Visser et al 2016 Methods Appl. Fluoresc. 4 035002). To this end, we used a general-purpose software module of MEM that was earlier developed to analyze (complex) laser photolysis kinetics of ligand rebinding reactions in oxygen binding proteins. We demonstrate that the MEM software provides reliable results and is easy to use for the analysis of both total fluorescence decay and fluorescence anisotropy decay of aqueous solutions of EGFP. The rotational correlation times of EGFP in water/glycerol mixtures, obtained by MEM as maxima of the correlation-time distributions, are identical to the single correlation times determined by global analysis of parallel and perpendicular polarized decay components. The MEM software is also able to determine homo-FRET in another dimeric GFP, for which the transfer correlation time is an order of magnitude shorter than the rotational correlation time. One important advantage utilizing MEM analysis is that no initial guesses of parameters are required, since MEM is able to select the least correlated solution from the feasible set of solutions.

Differentiation of ocular fundus fluorophores by fluorescence lifetime imaging using multiple excitation and emission wavelengths

NASA Astrophysics Data System (ADS)

Hammer, M.; Schweitzer, D.; Schenke, S.; Becker, W.; Bergmann, A.

2006-10-01

Ocular fundus autofluorescence imaging has been introduced into clinical diagnostics recently. It is in use for the observation of the age pigment lipofuscin, a precursor of age - related macular degeneration (AMD). But other fluorophores may be of interest too: The redox pair FAD - FADH II provides information on the retinal energy metabolism, advanced glycation end products (AGE) indicate protein glycation associated with pathologic processes in diabetes as well as AMD, and alterations in the fluorescence of collagen and elastin in connective tissue give us the opportunity to observe fibrosis by fluorescence imaging. This, however, needs techniques able to differentiate particular fluorophores despite limited permissible ocular exposure as well as excitation wavelength (limited by the transmission of the human ocular lens to >400 nm). We present an ophthalmic laser scanning system (SLO), equipped with picosecond laser diodes (FWHM 100 ps, 446 nm or 468 nm respectively) and time correlated single photon counting (TCSPC) in two emission bands (500 - 560 nm and 560 - 700 nm). The decays were fitted by a bi-exponential model. Fluorescence spectra were measured by a fluorescence spectrometer fluorolog. Upon excitation at 446 nm, the fluorescence of AGE, FAD, and lipofuscin were found to peak at 503 nm, 525 nm, and 600 nm respectively. Accordingly, the statistical distribution of the fluorescence decay times was found to depend on the different excitation wavelengths and emission bands used. The use of multiple excitation and emission wavelengths in conjunction with fluorescence lifetime imaging allows us to discriminate between intrinsic fluorophores of the ocular fundus. Taken together with our knowledge on the anatomical structure of the fundus, these findings suggest an association of the short, middle and long fluorescence decay time to the retinal pigment epithelium, the retina, and connective tissue respectively.

Real-time visualization of melanin granules in normal human skin using combined multiphoton and reflectance confocal microscopy.

PubMed

Majdzadeh, Ali; Lee, Anthony M D; Wang, Hequn; Lui, Harvey; McLean, David I; Crawford, Richard I; Zloty, David; Zeng, Haishan

2015-05-01

Recent advances in biomedical optics have enabled dermal and epidermal components to be visualized at subcellular resolution and assessed noninvasively. Multiphoton microscopy (MPM) and reflectance confocal microscopy (RCM) are noninvasive imaging modalities that have demonstrated promising results in imaging skin micromorphology, and which provide complementary information regarding skin components. This study assesses whether combined MPM/RCM can visualize intracellular and extracellular melanin granules in the epidermis and dermis of normal human skin. We perform MPM and RCM imaging of in vivo and ex vivo skin in the infrared domain. The inherent three-dimensional optical sectioning capability of MPM/RCM is used to image high-contrast granular features across skin depths ranging from 50 to 90 μm. The optical images thus obtained were correlated with conventional histologic examination including melanin-specific staining of ex vivo specimens. MPM revealed highly fluorescent granular structures below the dermal-epidermal junction (DEJ) region. Histochemical staining also demonstrated melanin-containing granules that correlate well in size and location with the granular fluorescent structures observed in MPM. Furthermore, the MPM fluorescence excitation wavelength and RCM reflectance of cell culture-derived melanin were equivalent to those of the granules. This study suggests that MPM can noninvasively visualize and quantify subepidermal melanin in situ. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Advances in quantitative UV-visible spectroscopy for clinical and pre-clinical application in cancer.

PubMed

Brown, J Quincy; Vishwanath, Karthik; Palmer, Gregory M; Ramanujam, Nirmala

2009-02-01

Methods of optical spectroscopy that provide quantitative, physically or physiologically meaningful measures of tissue properties are an attractive tool for the study, diagnosis, prognosis, and treatment of various cancers. Recent development of methodologies to convert measured reflectance and fluorescence spectra from tissue to cancer-relevant parameters such as vascular volume, oxygenation, extracellular matrix extent, metabolic redox states, and cellular proliferation have significantly advanced the field of tissue optical spectroscopy. The number of publications reporting quantitative tissue spectroscopy results in the UV-visible wavelength range has increased sharply in the past three years, and includes new and emerging studies that correlate optically measured parameters with independent measures such as immunohistochemistry, which should aid in increased clinical acceptance of these technologies.

Importing super-resolution imaging into nanoscale puzzles of materials dynamics

NASA Astrophysics Data System (ADS)

King, John; Tsang, Chi Hang Boyce; Wilson, William; Granick, Steve

2014-03-01

A limitation of the exciting recent advances in sub-diffraction microscopy is that they focus on imaging rather than dynamical changes. We are engaged in extending this technique beyond the usual biological applications to address materials problems instead. To this end, we employ stimulated emission depletion (STED) microscopy, which relies on selectively turning off fluorescence emitters through stimulated emission, allowing only a small subset of emitters to be detected, such that the excitation spot size can be downsized to tens of nanometers. By coupling the STED excitation scheme to fluorescence correlation spectroscopy (FCS), diffusive processes are studied with nanoscale resolution. Here, we demonstrate the benefits of such experimental capabilities in a diverse range of complex systems, ranging from the diffusion of nano-objects in crowded 3D environments to the study of polymer diffusion on 2D surfaces.

Fluorescence correlation spectroscopy of diffusion probed with a Gaussian Lorentzian spatial distribution

NASA Astrophysics Data System (ADS)

Marrocco, Michele

2007-11-01

Fluorescence correlation spectroscopy is fundamental in many physical, chemical and biological studies of molecular diffusion. However, the concept of fluorescence correlation is founded on the assumption that the analytical description of the correlation decay of diffusion can be achieved if the spatial profile of the detected volume obeys a three-dimensional Gaussian distribution. In the present Letter, the analytical result is instead proven for the fundamental Gaussian-Lorentzian profile.

Advanced Methods in Fluorescence Microscopy

PubMed Central

Fritzky, Luke; Lagunoff, David

2013-01-01

It requires a good deal of will power to resist hyperbole in considering the advances that have been achieved in fluorescence microscopy in the last 25 years. Our effort has been to survey the modalities of microscopic fluorescence imaging available to cell biologists and perhaps useful for diagnostic pathologists. The gamut extends from established confocal laser scanning through multiphoton and TIRF to the emerging technologies of super-resolution microscopy that breech the Abbé limit of resolution. Also considered are the recent innovations in structured and light sheet illumination, the use of FRET and molecular beacons that exploit specific characteristics of designer fluorescent proteins, fluorescence speckles, and second harmonic generation for native anisometric structures like collagen, microtubules and sarcomeres. PMID:23271142

Advanced methods in fluorescence microscopy.

PubMed

Fritzky, Luke; Lagunoff, David

2013-01-01

It requires a good deal of will power to resist hyperbole in considering the advances that have been achieved in fluorescence microscopy in the last 25 years. Our effort has been to survey the modalities of microscopic fluorescence imaging available to cell biologists and perhaps useful for diagnostic pathologists. The gamut extends from established confocal laser scanning through multiphoton and TIRF to the emerging technologies of super-resolution microscopy that breech the Abbe limit of resolution. Also considered are the recent innovations in structured and light sheet illumination, the use of FRET and molecular beacons that exploit specific characteristics of designer fluorescent proteins, fluorescence speckles, and second harmonic generation for native anisometric structures like collagen, microtubules and sarcomeres.

Advanced methods in fluorescence microscopy.

PubMed

Fritzky, Luke; Lagunoff, David

2013-01-01

It requires a good deal of will power to resist hyperbole in considering the advances that have been achieved in fluorescence microscopy in the last 25 years. Our effort has been to survey the modalities of microscopic fluorescence imaging available to cell biologists and perhaps useful for diagnostic pathologists. The gamut extends from established confocal laser scanning through multiphoton and TIRF to the emerging technologies of super-resolution microscopy that breech the Abbé limit of resolution. Also considered are the recent innovations in structured and light sheet illumination, the use of FRET and molecular beacons that exploit specific characteristics of designer fluorescent proteins, fluorescence speckles, and second harmonic generation for native anisometric structures like collagen, microtubules and sarcomeres.

Monitoring the Behavior of Emerging Contaminants in Wastewater-Impacted Rivers Based on the Use of Fluorescence Excitation Emission Matrixes (EEM).

PubMed

Sgroi, Massimiliano; Roccaro, Paolo; Korshin, Gregory V; Vagliasindi, Federico G A

2017-04-18

This study investigated the applicability of fluorescence indexes based on the interpretation of excitation emission matrices (EEMs) by PARAFAC analysis and by selecting fluorescence intensities at a priori defined excitation/emission pairs as surrogates for monitoring the behavior of emerging organic compounds (EOCs) in two catchment basins impacted by wastewater discharges. Relevant EOC and EEM data were obtained for a 90 km stretch of the Simeto River, the main river in Sicily, and the smaller San Leonardo River, which was investigated for a 17 km stretch. The use of fluorescence indexes developed by these two different approaches resulted in similar observations. Changes of the fluorescence indexes that correspond to a group of humic-like fluorescing species were determined to be highly correlated with the concentrations of recalcitrant contaminants such as sucralose, sulfamethoxazole and carbamazepine, which are typical wastewater markers in river water. Changes of the fluorescence indexes related to tyrosine-like substances were well correlated with the concentrations of ibuprofen and caffeine, anthropogenic indicators of untreated wastewater discharges. Chemical oxygen demand and dissolved organic carbon concentrations were correlated with humic-like fluorescence indexes. The observed correlations were site-specific and characterized by different regression parameters for every collection event. Caffeine and carbamazepine showed correlations with florescence indexes in the San Leonardo River and in the alluvial plain stretch of the Simeto River, whereas sucralose, sulfamethoxazole and ibuprofen have always been well correlated in all the investigated river stretches. However, when data of different collection events from river stretches where correlations were observed were combined, good linear correlations were obtained for data sets generated via the normalization of the measured concentrations by the average value for the corresponding collection event. These results show that fluorescence based indexes can be used to monitor the behavior of some trace organic contaminants in wastewater impacted rivers and to track wastewater discharges in streams and rivers.

Correlative Stochastic Optical Reconstruction Microscopy and Electron Microscopy

PubMed Central

Kim, Doory; Deerinck, Thomas J.; Sigal, Yaron M.; Babcock, Hazen P.; Ellisman, Mark H.; Zhuang, Xiaowei

2015-01-01

Correlative fluorescence light microscopy and electron microscopy allows the imaging of spatial distributions of specific biomolecules in the context of cellular ultrastructure. Recent development of super-resolution fluorescence microscopy allows the location of molecules to be determined with nanometer-scale spatial resolution. However, correlative super-resolution fluorescence microscopy and electron microscopy (EM) still remains challenging because the optimal specimen preparation and imaging conditions for super-resolution fluorescence microscopy and EM are often not compatible. Here, we have developed several experiment protocols for correlative stochastic optical reconstruction microscopy (STORM) and EM methods, both for un-embedded samples by applying EM-specific sample preparations after STORM imaging and for embedded and sectioned samples by optimizing the fluorescence under EM fixation, staining and embedding conditions. We demonstrated these methods using a variety of cellular targets. PMID:25874453

Correlation fluorescence method of amine detection

NASA Astrophysics Data System (ADS)

Myslitsky, Valentin F.; Tkachuk, Svetlana S.; Rudeichuk, Volodimir M.; Strinadko, Miroslav T.; Slyotov, Mikhail M.; Strinadko, Marina M.

1997-12-01

The amines fluorescence spectra stimulated by UV laser radiation are investigated in this paper. The fluorescence is stimulated by the coherent laser beam with the wavelength 0.337 micrometers . At the sufficient energy of laser stimulation the narrow peaks of the fluorescence spectra are detected besides the wide maximum. The relationship between the fluorescence intensity and the concentration of amines solutions are investigated. The fluorescence intensity temporal dependence on wavelength 0.363 micrometers of the norepinephrine solution preliminarily radiated by UV laser with wavelength 0.337 micrometers was found. The computer stimulated and experimental investigations of adrenaline and norepinephrine mixtures fluorescence spectra were done. The correlation fluorescent method of amines detection is proposed.

Fluorescence lifetime assays: current advances and applications in drug discovery.

PubMed

Pritz, Stephan; Doering, Klaus; Woelcke, Julian; Hassiepen, Ulrich

2011-06-01

Fluorescence lifetime assays complement the portfolio of established assay formats available in drug discovery, particularly with the recent advances in microplate readers and the commercial availability of novel fluorescent labels. Fluorescence lifetime assists in lowering complexity of compound screening assays, affording a modular, toolbox-like approach to assay development and yielding robust homogeneous assays. To date, materials and procedures have been reported for biochemical assays on proteases, as well as on protein kinases and phosphatases. This article gives an overview of two assay families, distinguished by the origin of the fluorescence signal modulation. The pharmaceutical industry demands techniques with a robust, integrated compound profiling process and short turnaround times. Fluorescence lifetime assays have already helped the drug discovery field, in this sense, by enhancing productivity during the hit-to-lead and lead optimization phases. Future work will focus on covering other biochemical molecular modifications by investigating the detailed photo-physical mechanisms underlying the fluorescence signal.

Advanced fluorescence microscopy techniques for the life sciences

PubMed Central

Aguib, Yasmine; Yacoub, Magdi H.

The development of super-resolved fluorescence microscopy, for which the Nobel Prize was awarded in 2014, has been a topic of interest to physicists and biologists alike. It is inevitable that numerous questions in biomedical research cannot be answered by means other than direct observation. In this review, advances to fluorescence microscopy are covered in a widely accessible fashion to facilitate its use in decisions related to its acquisition and utilization in biomedical research. PMID:29043264

Prediction of anaerobic biodegradability and bioaccessibility of municipal sludge by coupling sequential extractions with fluorescence spectroscopy: towards ADM1 variables characterization.

PubMed

Jimenez, Julie; Gonidec, Estelle; Cacho Rivero, Jesús Andrés; Latrille, Eric; Vedrenne, Fabien; Steyer, Jean-Philippe

2014-03-01

Advanced dynamic anaerobic digestion models, such as ADM1, require both detailed organic matter characterisation and intimate knowledge of the involved metabolic pathways. In the current study, a methodology for municipal sludge characterization is investigated to describe two key parameters: biodegradability and bioaccessibility of organic matter. The methodology is based on coupling sequential chemical extractions with 3D fluorescence spectroscopy. The use of increasingly strong solvents reveals different levels of organic matter accessibility and the spectroscopy measurement leads to a detailed characterisation of the organic matter. The results obtained from testing 52 municipal sludge samples (primary, secondary, digested and thermally treated) showed a successful correlation with sludge biodegradability and bioaccessibility. The two parameters, traditionally obtained through the biochemical methane potential (BMP) lab tests, are now obtain in only 5 days compared to the 30-60 days usually required. Experimental data, obtained from two different laboratory scale reactors, were used to validate the ADM1 model. The proposed approach showed a strong application potential for reactor design and advanced control of anaerobic digestion processes. Copyright © 2013 Elsevier Ltd. All rights reserved.

Portable fluorescence meter with reference backscattering channel

NASA Astrophysics Data System (ADS)

Kornilin, Dmitriy V.; Grishanov, Vladimir N.; Zakharov, Valery P.; Burkov, Dmitriy S.

2016-09-01

Methods based on fluorescence and backscattering are intensively used for determination of the advanced glycation end products (AGE) concentration in the biological tissues. There are strong correlation between the AGE concentration and the severity of such diseases like diabetes, coronary heart disease and renal failure. This fact can be used for diagnostic purposes in medical applications. Only few investigations in this area can be useful for development of portable and affordable in vivo AGE meter because the most of them are oriented on using spectrometers. In this study we describe the design and the results of tests on volunteers of portable fluorescence meter based on two photodiodes. One channel of such fluorimeter is used for measurement of the autofluorescence (AF) intensity, another one - for the intensity of elastically scattered radiation, which can be used as a reference. This reference channel is proposed for normalization of the skin autofluorescence signal to the human skin photo type. The fluorimeter, that was developed is relatively compact and does not contain any expensive optical and electronic components. The experimental results prove that proposed tool can be used for the AGE estimation in human skin.

Fluorescence-based visualization of autophagic activity predicts mouse embryo viability

NASA Astrophysics Data System (ADS)

Tsukamoto, Satoshi; Hara, Taichi; Yamamoto, Atsushi; Kito, Seiji; Minami, Naojiro; Kubota, Toshiro; Sato, Ken; Kokubo, Toshiaki

2014-03-01

Embryo quality is a critical parameter in assisted reproductive technologies. Although embryo quality can be evaluated morphologically, embryo morphology does not correlate perfectly with embryo viability. To improve this, it is important to understand which molecular mechanisms are involved in embryo quality control. Autophagy is an evolutionarily conserved catabolic process in which cytoplasmic materials sequestered by autophagosomes are degraded in lysosomes. We previously demonstrated that autophagy is highly activated after fertilization and is essential for further embryonic development. Here, we developed a simple fluorescence-based method for visualizing autophagic activity in live mouse embryos. Our method is based on imaging of the fluorescence intensity of GFP-LC3, a versatile marker for autophagy, which is microinjected into the embryos. Using this method, we show that embryonic autophagic activity declines with advancing maternal age, probably due to a decline in the activity of lysosomal hydrolases. We also demonstrate that embryonic autophagic activity is associated with the developmental viability of the embryo. Our results suggest that embryonic autophagic activity can be utilized as a novel indicator of embryo quality.

Dual-color two-photon fluorescence correlation spectroscopy

NASA Astrophysics Data System (ADS)

Berland, Keith M.

2001-04-01

Fluorescence correlation spectroscopy (FCS) is rapidly growing in popularity as a research tool in biological and biophysical research. Under favorable conditions, FCS measurements can produce an accurate characterization of the chemical, physical, and kinetic properties of a biological system. However, interpretation of FCS data quickly becomes complicated as the heterogeneity of a molecular system increases, as well as when there is significant non-stationery fluorescence background (e.g. intracellular autofluorescence). Use of multi-parameter correlation measurements is one promising approach that can improve the fidelity of FCS measurements in complex systems. In particular, the use of dual-color fluorescence assays, in which different interacting molecular species are labeled with unique fluorescent indicators, can "tune" the sensitivity of FCS measurements in favor of particular molecular species of interest, while simultaneously minimizing the contribution of other molecular species to the overall fluorescence correlation signal. Here we introduce the combined application of two-photon fluorescence excitation and dual-color cross-correlation analysis for detecting molecular interactions in solution. The use of two-photon excitation is particularly advantageous for dual-color FCS applications due to the uncomplicated optical alignment and the superior capabilities for intracellular applications. The theory of two-photon dual-color FCS is introduced, and initial results quantifying hybridization reactions between three independent single stranded DNA molecules are presented.

Enhancing the sensitivity of fluorescence correlation spectroscopy by using time-correlated single photon counting.

PubMed

Lamb, D C; Müller, B K; Bräuchle, C

2005-10-01

Fluorescence correlation spectroscopy (FCS) and fluorescence cross-correlation spectroscopy (FCCS) are methods that extract information about a sample from the influence of thermodynamic equilibrium fluctuations on the fluorescence intensity. This method allows dynamic information to be obtained from steady state equilibrium measurements and its popularity has dramatically increased in the last 10 years due to the development of high sensitivity detectors and its combination with confocal microscopy. Using time-correlated single-photon counting (TCSPC) detection and pulsed excitation, information over the duration of the excited state can be extracted and incorporated in the analysis. In this short review, we discuss new methodologies that have recently emerged which incorporated fluorescence lifetime information or TCSPC data in the FCS and FCCS analysis. Time-gated FCS discriminates between which photons are to be incorporated in the analysis dependent upon their arrival time after excitation. This allows for accurate FCS measurements in the presence of fluorescent background, determination of sample homogeneity, and the ability to distinguish between static and dynamic heterogeneities. A similar method, time-resolved FCS can be used to resolve the individual correlation functions from multiple fluorophores through the different fluorescence lifetimes. Pulsed interleaved excitation (PIE) encodes the excitation source into the TCSPC data. PIE can be used to perform dual-channel FCCS with a single detector and allows elimination of spectral cross-talk with dual-channel detection. For samples that undergo fluorescence resonance energy transfer (FRET), quantitative FCCS measurements can be performed in spite of the FRET and the static FRET efficiency can be determined.

Simple non-invasive assessment of advanced glycation endproduct accumulation.

PubMed

Meerwaldt, R; Graaff, R; Oomen, P H N; Links, T P; Jager, J J; Alderson, N L; Thorpe, S R; Baynes, J W; Gans, R O B; Smit, A J

2004-07-01

The accumulation of AGE is thought to play a role in the pathogenesis of chronic complications of diabetes mellitus and renal failure. All current measurements of AGE accumulation require invasive sampling. We exploited the fact that several AGE exhibit autofluorescence to develop a non-invasive tool for measuring skin AGE accumulation, the Autofluorescence Reader (AFR). We validated its use by comparing the values obtained using the AFR with the AGE content measured in extracts from skin biopsies of diabetic and control subjects. Using the AFR with an excitation light source of 300-420 nm, fluorescence of the skin was measured at the arm and lower leg in 46 patients with diabetes (Type 1 and 2) and in 46 age- and sex-matched control subjects, the majority of whom were Caucasian. Autofluorescence was defined as the average fluorescence per nm over the entire emission spectrum (420-600 nm) as ratio of the average fluorescence per nm over the 300-420-nm range. Skin biopsies were obtained from the same site of the arm, and analysed for collagen-linked fluorescence (CLF) and specific AGE: pentosidine, N(epsilon)-(carboxymethyl)lysine (CML) and N(epsilon)-(carboxyethyl)lysine (CEL). Autofluorescence correlated with CLF, pentosidine, CML, and CEL ( r=0.47-0.62, p

Drought is Coming: Monitoring Vegetation Response to Water Scarcity through Variable Chlorophyll a Fluorescence

NASA Astrophysics Data System (ADS)

Guadagno, C. R.; Beverly, D.; Pleban, J. R.; Speckman, H. N.; Ewers, B. E.; Weinig, C.

2017-12-01

Aridity is one of the most pronounced environmental limits to plant survival, and understanding how plants respond to drought and recovery is crucial for predicting impacts on managed and natural ecosystems. Changes in soil moisture conditions induce a suite of physiological responses from the cell to ecosystem scale, complicating the assessment of drought effects. Characterizing early indicators of water scarcity across species can inform biophysical models with improved understanding of plant hydraulics. While indexes exist for drought monitoring across scales, many are unable to identify imminent vegetative drought. We explore a method of early diagnosis using leaf-level and kinetic imaging measures of variable chlorophyll a fluorescence. This is a fast and reliable tool capturing leaf physiological changes in advance of changes in NDVI or passive solar induced fluorescence. Both image and leaf level Pulse Amplitude Method (PAM) measurements illustrate the utility of variable chlorophyll a fluorescence for monitoring vegetative drought. Variable fluorescence was monitored across populations of crops, desert shrubs, montane conifers and riparian deciduous trees under variable water regimes. We found a strong correlation (R = 0.85) between the maximum efficiency of photosystem II measured using variable fluorescence (Fv'Fm') and leaf level electrolyte leakage, a proximal cause of drought stress induced by cellular damage in leaves. This association was confirmed in two gymnosperm species (Picea engelmannii and Pinus contorta) and for diverse varieties of the crop species Brassica rapa. The use of chlorophyll a fluorescence per image also allowed for early detection of drought in aspen (Populus tremuloides). These results provide evidence that variable chlorophyll fluorescence decreases between 25% and 70% in mild and severely droughted twigs with respect to ones collected from trees in wet soil conditions. While current systems for monitoring variable fluorescence are limited in scale, chlorophyll fluorescence comprises an indicator of drought stress across multiple spatial scales, from leaf to ecosystem level.

Diffusion Tensor Analysis by Two-Dimensional Pair Correlation of Fluorescence Fluctuations in Cells.

PubMed

Di Rienzo, Carmine; Cardarelli, Francesco; Di Luca, Mariagrazia; Beltram, Fabio; Gratton, Enrico

2016-08-23

In a living cell, the movement of biomolecules is highly regulated by the cellular organization into subcompartments that impose barriers to diffusion, can locally break the spatial isotropy, and ultimately guide these molecules to their targets. Despite the pivotal role of these processes, experimental tools to fully probe the complex connectivity (and accessibility) of the cell interior with adequate spatiotemporal resolution are still lacking. Here, we show how the heterogeneity of molecular dynamics and the location of barriers to molecular motion can be mapped in live cells by exploiting a two-dimensional (2D) extension of the pair correlation function (pCF) analysis. Starting from a time series of images collected for the same field of view, the resulting 2D pCF is calculated in the proximity of each point for each time delay and allows us to probe the spatial distribution of the molecules that started from a given pixel. This 2D pCF yields an accurate description of the preferential diffusive routes. Furthermore, we combine this analysis with the image-derived mean-square displacement approach and gain information on the average nanoscopic molecular displacements in different directions. Through these quantities, we build a fluorescence-fluctuation-based diffusion tensor that contains information on speed and directionality of the local dynamical processes. Contrary to classical fluorescence correlation spectroscopy and related methods, this combined approach can distinguish between isotropic and anisotropic local diffusion. We argue that the measurement of this iMSD tensor will contribute to advance our understanding of the role played by the intracellular environment in the regulation of molecular diffusion at the nanoscale. Copyright © 2016 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Fluorescence lifetime correlation spectroscopy for precise concentration detection in vivo by background subtraction

NASA Astrophysics Data System (ADS)

Gärtner, Maria; Mütze, Jörg; Ohrt, Thomas; Schwille, Petra

2009-07-01

In vivo studies of single molecule dynamics by means of Fluorescence correlation spectroscopy can suffer from high background. Fluorescence lifetime correlation spectroscopy provides a tool to distinguish between signal and unwanted contributions via lifetime separation. By studying the motion of the RNA-induced silencing complex (RISC) within two compartments of a human cell, the nucleus and the cytoplasm, we observed clear differences in concentration as well as mobility of the protein complex between those two locations. Especially in the nucleus, where the fluorescence signal is very weak, a correction for background is crucial to provide reliable results of the particle number. Utilizing the fluorescent lifetime of the different contributions, we show that it is possible to distinguish between the fluorescent signal and the autofluorescent background in vivo in a single measurement.

Discerning the Chemistry in Individual Organelles with Small-Molecule Fluorescent Probes.

PubMed

Xu, Wang; Zeng, Zebing; Jiang, Jian-Hui; Chang, Young-Tae; Yuan, Lin

2016-10-24

Principle has it that even the most advanced super-resolution microscope would be futile in providing biological insight into subcellular matrices without well-designed fluorescent tags/probes. Developments in biology have increasingly been boosted by advances of chemistry, with one prominent example being small-molecule fluorescent probes that not only allow cellular-level imaging, but also subcellular imaging. A majority, if not all, of the chemical/biological events take place inside cellular organelles, and researchers have been shifting their attention towards these substructures with the help of fluorescence techniques. This Review summarizes the existing fluorescent probes that target chemical/biological events within a single organelle. More importantly, organelle-anchoring strategies are described and emphasized to inspire the design of new generations of fluorescent probes, before concluding with future prospects on the possible further development of chemical biology. © 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Feasibility of the simultaneous determination of polycyclic aromatic hydrocarbons based on two-dimensional fluorescence correlation spectroscopy

NASA Astrophysics Data System (ADS)

Yang, Renjie; Dong, Guimei; Sun, Xueshan; Yang, Yanrong; Yu, Yaping; Liu, Haixue; Zhang, Weiyu

2018-02-01

A new approach for quantitative determination of polycyclic aromatic hydrocarbons (PAHs) in environment was proposed based on two-dimensional (2D) fluorescence correlation spectroscopy in conjunction with multivariate method. 40 mixture solutions of anthracene and pyrene were prepared in the laboratory. Excitation-emission matrix (EEM) fluorescence spectra of all samples were collected. And 2D fluorescence correlation spectra were calculated under the excitation perturbation. The N-way partial least squares (N-PLS) models were developed based on 2D fluorescence correlation spectra, showing a root mean square error of calibration (RMSEC) of 3.50 μg L- 1 and root mean square error of prediction (RMSEP) of 4.42 μg L- 1 for anthracene and of 3.61 μg L- 1 and 4.29 μg L- 1 for pyrene, respectively. Also, the N-PLS models were developed for quantitative analysis of anthracene and pyrene using EEM fluorescence spectra. The RMSEC and RMSEP were 3.97 μg L- 1 and 4.63 μg L- 1 for anthracene, 4.46 μg L- 1 and 4.52 μg L- 1 for pyrene, respectively. It was found that the N-PLS model using 2D fluorescence correlation spectra could provide better results comparing with EEM fluorescence spectra because of its low RMSEC and RMSEP. The methodology proposed has the potential to be an alternative method for detection of PAHs in environment.

Zebrafish Caudal Fin Angiogenesis Assay—Advanced Quantitative Assessment Including 3-Way Correlative Microscopy

PubMed Central

Correa Shokiche, Carlos; Schaad, Laura; Triet, Ramona; Jazwinska, Anna; Tschanz, Stefan A.; Djonov, Valentin

2016-01-01

Background Researchers evaluating angiomodulating compounds as a part of scientific projects or pre-clinical studies are often confronted with limitations of applied animal models. The rough and insufficient early-stage compound assessment without reliable quantification of the vascular response counts, at least partially, to the low transition rate to clinics. Objective To establish an advanced, rapid and cost-effective angiogenesis assay for the precise and sensitive assessment of angiomodulating compounds using zebrafish caudal fin regeneration. It should provide information regarding the angiogenic mechanisms involved and should include qualitative and quantitative data of drug effects in a non-biased and time-efficient way. Approach & Results Basic vascular parameters (total regenerated area, vascular projection area, contour length, vessel area density) were extracted from in vivo fluorescence microscopy images using a stereological approach. Skeletonization of the vasculature by our custom-made software Skelios provided additional parameters including “graph energy” and “distance to farthest node”. The latter gave important insights into the complexity, connectivity and maturation status of the regenerating vascular network. The employment of a reference point (vascular parameters prior amputation) is unique for the model and crucial for a proper assessment. Additionally, the assay provides exceptional possibilities for correlative microscopy by combining in vivo-imaging and morphological investigation of the area of interest. The 3-way correlative microscopy links the dynamic changes in vivo with their structural substrate at the subcellular level. Conclusions The improved zebrafish fin regeneration model with advanced quantitative analysis and optional 3-way correlative morphology is a promising in vivo angiogenesis assay, well-suitable for basic research and preclinical investigations. PMID:26950851

Contribution of high-resolution correlative imaging techniques in the study of the liver sieve in three-dimensions.

PubMed

Braet, Filip; Wisse, Eddie; Bomans, Paul; Frederik, Peter; Geerts, Willie; Koster, Abraham; Soon, Lilian; Ringer, Simon

2007-03-01

Correlative microscopy has become increasingly important for the analysis of the structure, function, and dynamics of cells. This is largely due to the result of recent advances in light-, probe-, laser- and various electron microscopy techniques that facilitate three-dimensional studies. Furthermore, the improved understanding in the past decade of imaging cell compartments in the third dimension has resulted largely from the availability of powerful computers, fast high-resolution CCD cameras, specifically developed imaging analysis software, and various probes designed for labeling living and or fixed cells. In this paper, we review different correlative high-resolution imaging methodologies and how these microscopy techniques facilitated the accumulation of new insights in the morpho-functional and structural organization of the hepatic sieve. Various aspects of hepatic endothelial fenestrae regarding their structure, origin, dynamics, and formation will be explored throughout this paper by comparing the results of confocal laser scanning-, correlative fluorescence and scanning electron-, atomic force-, and whole-mount electron microscopy. Furthermore, the recent advances of vitrifying cells with the vitrobot in combination with the glove box for the preparation of cells for cryo-electron microscopic investigation will be discussed. Finally, the first transmission electron tomography data of the liver sieve in three-dimensions are presented. The obtained data unambiguously show the involvement of special domains in the de novo formation and disappearance of hepatic fenestrae, and focuses future research into the (supra)molecular structure of the fenestrae-forming center, defenestration center and fenestrae-, and sieve plate cytoskeleton ring by using advanced cryo-electron tomography. (c) 2007 Wiley-Liss, Inc.

A total internal reflection-fluorescence correlation spectroscopy setup with pulsed diode laser excitation

NASA Astrophysics Data System (ADS)

Weger, Lukas; Hoffmann-Jacobsen, Kerstin

2017-09-01

Fluorescence correlation spectroscopy (FCS) measures fluctuations in a (sub-)femtoliter volume to analyze the diffusive behavior of fluorescent particles. This highly sensitive method has proven to be useful for the analysis of dynamic biological systems as well as in chemistry, physics, and material sciences. It is routinely performed with commercial fluorescence microscopes, which provide a confined observation volume by the confocal technique. The evanescent wave of total internal reflectance (TIR) is used in home-built systems to permit a surface sensitive FCS analysis. We present a combined confocal and TIR-FCS setup which uses economic low-power pulsed diode lasers for excitation. Excitation and detection are coupled to time-correlated photon counting hardware. This allows simultaneous fluorescence lifetime and FCS measurements in a surface-sensitive mode. Moreover, the setup supports fluorescence lifetime correlation spectroscopy at surfaces. The excitation can be easily switched between TIR and epi-illumination to compare the surface properties with those in liquid bulk. The capabilities of the presented setup are demonstrated by measuring the diffusion coefficients of a free dye molecule, a labeled polyethylene glycol, and a fluorescent nanoparticle in confocal as well as in TIR-FCS.

Simple noninvasive measurement of skin autofluorescence.

PubMed

Meerwaldt, Robbert; Links, Thera; Graaff, Reindert; Thorpe, Suzannne R; Baynes, John W; Hartog, Jasper; Gans, Reinold; Smit, Andries

2005-06-01

Accumulation of advanced glycation end products (AGEs) is thought to play a role in the pathogenesis of chronic complications of diabetes mellitus and renal failure. Several studies indicate that AGE accumulation in tissue may reflect the cumulative effect of hyperglycemia and oxidative stress over many years. Simple quantitation of AGE accumulation in tissue could provide a tool for assessing the risk of long-term complications. Because several AGEs exhibit autofluorescence, we developed a noninvasive autofluorescence reader (AFR). Skin autofluorescence measured with the AFR correlates with collagen-linked fluorescence and specific skin AGE levels from skin biopsy samples. Furthermore, skin autofluorescence correlates with long-term glycemic control and renal function, and preliminary results show correlations with the presence of long-term complications in diabetes. The AFR may be useful as a clinical tool for rapid assessment of risk for AGE-related long-term complications in diabetes and in other conditions associated with AGE accumulation.

Fluorescence Spectroscopy as a Tool for the Assessment of Liver Samples with Several Stages of Fibrosis.

PubMed

Fabila-Bustos, Diego A; Arroyo-Camarena, Úrsula D; López-Vancell, María D; Durán-Padilla, Marco A; Azuceno-García, Itzel; Stolik-Isakina, Suren; Valor-Reed, Alma; Ibarra-Coronado, Elizabeth; Hernández-Quintanar, Luis F; Escobedo, Galileo; de la Rosa-Vázquez, José M

2018-03-01

During the last years, fluorescence spectroscopy has been used as a potential tool for the evaluation and characterization of tissues with different disease conditions due to its low cost, high sensitivity, and minimally or noninvasive character. In this study, fluorescence spectroscopy was used to study 19 paraffin blocks containing human liver tissue from biopsies. All samples were previously analyzed by two senior pathologists in a single-blind trial. After their evaluation, four liver samples were classified as nonfibrosis (F0), four as initial fibrosis (F1-F2), four as advanced fibrosis (F3), and six as cirrhosis (F4). The fluorescence was induced at different wavelengths as follows: 330, 365, and 405 nm using a portable fiber-optic system. The fluorescence spectra were recorded in the range of 400-750 nm. A distinctive correlation between the shape of each spectrum and the level of fibrosis in the liver sample was detected. A multi-variate statistical analysis based on principal component analysis followed by linear discrimination analysis was applied to develop algorithms able to distinguish different stages of fibrosis based on the characteristics of fluorescence spectra. Pairwise comparisons were performed: F0 versus F1-F2, F1-F2 versus F3, F3 versus F4, and F1-F2 versus F4. The algorithms applied to each set of data yielded values of sensitivity and specificity that were higher than 90% and 95%, respectively, in all the analyzed cases. With this study, it is concluded that fluorescence spectroscopy can be used as a complementary tool for the assessment of liver fibrosis in liver tissue samples, which sets the stage for subsequent clinical trials.

Fluorescence-based multi-parameter approach to characterize dynamics of organic carbon, faecal bacteria and particles at alpine karst springs.

PubMed

Frank, Simon; Goeppert, Nadine; Goldscheider, Nico

2018-02-15

Karst springs, especially in alpine regions, are important for drinking water supply but also vulnerable to contamination, especially after rainfall events. This high variability of water quality requires rapid quantification of contamination parameters. Here, we used a fluorescence-based multi-parameter approach to characterize the dynamics of organic carbon, faecal bacteria, and particles at three alpine karst springs. We used excitation emission matrices (EEMs) to identify fluorescent dissolved organic material (FDOM). At the first system, peak A fluorescence and total organic carbon (TOC) were strongly correlated (Spearman's r s of 0.949), indicating that a large part of the organic matter is related to humic-like substances. Protein-like fluorescence and cultivation-based determination of coliform bacteria also had a significant correlation with r s =0.734, indicating that protein-like fluorescence is directly related to faecal pollution. At the second system, which has two spring outlets, the absolute values of all measured water-quality parameters were lower; there was a significant correlation between TOC and humic-like fluorescence (r s =0.588-0.689) but coliform bacteria and protein-like fluorescence at these two springs were not correlated. Additionally, there was a strong correlation (r s =0.571-0.647) between small particle fractions (1.0 and 2.0μm), a secondary turbidity peak and bacteria. At one of these springs, discharge was constant despite the reaction of all other parameters to the rainfall event. Our results demonstrated that i) all three springs showed fast and marked responses of all investigated water-quality parameters after rain events; ii) a constant discharge does not necessarily mean constant water quality; iii) at high contamination levels, protein-like fluorescence is a good indicator of bacterial contamination, while at low contamination levels no correlation between protein-like fluorescence and bacterial values was detected; and iv) a combination of fluorescence measurements and particle-size analysis is a promising approach for a rapid assessment of organic contamination, especially relative to time-consuming conventional bacterial determination methods. Copyright © 2017 Elsevier B.V. All rights reserved.

Understanding Fluorescence Measurements through a Guided-Inquiry and Discovery Experiment in Advanced Analytical Laboratory

ERIC Educational Resources Information Center

Wilczek-Vera, Grazyna; Salin, Eric Dunbar

2011-01-01

An experiment on fluorescence spectroscopy suitable for an advanced analytical laboratory is presented. Its conceptual development used a combination of the expository and discovery styles. The "learn-as-you-go" and direct "hands-on" methodology applied ensures an active role for a student in the process of visualization and discovery of concepts.…

Eco-friendly carbon-nanodot-based fluorescent paints for advanced photocatalytic systems

PubMed Central

Young Park, So; Uk Lee, Hyun; Lee, Young-Chul; Choi, Saehae; Hyun Cho, Dae; Sik Kim, Hee; Bang, Sunghee; Seo, Soonjoo; Chang Lee, Soon; Won, Jonghan; Son, Byung-Chul; Yang, Mino; Lee, Jouhahn

2015-01-01

Fluorescent carbon nanomaterials, especially zero-dimensional (0D) carbon nanodots (CDs), are widely used in broad biological and optoelectronic applications. CDs have unique characteristics such as strong fluorescence, biocompatibility, sun-light response, and capability of mass-production. Beyond the previous green CD obtained from harmful natural substances, we report a new type of fluid-based fluorescent CD paints (C-paints) derived from polyethylene glycol (PEG; via simple ultrasound irradiation at room temperatures) and produced in quantum yields of up to ~14%. Additionally, C-paints possess a strong, UV- and visible-light-responsive photoluminescent (PL) property. Most especially, C-paints, by incorporation into a photocatalytic system, show additional roles in the emission of fluorescent light for activation of TiO2 nanoparticles (NPs) and the resultant detoxification of most organic dyes, thus further enabling embarkation in advanced water purification. PMID:26201431

Eco-friendly carbon-nanodot-based fluorescent paints for advanced photocatalytic systems.

PubMed

Park, So Young; Lee, Hyun Uk; Lee, Young-Chul; Choi, Saehae; Cho, Dae Hyun; Kim, Hee Sik; Bang, Sunghee; Seo, Soonjoo; Lee, Soon Chang; Won, Jonghan; Son, Byung-Chul; Yang, Mino; Lee, Jouhahn

2015-07-23

Fluorescent carbon nanomaterials, especially zero-dimensional (0D) carbon nanodots (CDs), are widely used in broad biological and optoelectronic applications. CDs have unique characteristics such as strong fluorescence, biocompatibility, sun-light response, and capability of mass-production. Beyond the previous green CD obtained from harmful natural substances, we report a new type of fluid-based fluorescent CD paints (C-paints) derived from polyethylene glycol (PEG; via simple ultrasound irradiation at room temperatures) and produced in quantum yields of up to ~14%. Additionally, C-paints possess a strong, UV- and visible-light-responsive photoluminescent (PL) property. Most especially, C-paints, by incorporation into a photocatalytic system, show additional roles in the emission of fluorescent light for activation of TiO2 nanoparticles (NPs) and the resultant detoxification of most organic dyes, thus further enabling embarkation in advanced water purification.

Correlative Super-Resolution Microscopy: New Dimensions and New Opportunities.

PubMed

Hauser, Meghan; Wojcik, Michal; Kim, Doory; Mahmoudi, Morteza; Li, Wan; Xu, Ke

2017-06-14

Correlative microscopy, the integration of two or more microscopy techniques performed on the same sample, produces results that emphasize the strengths of each technique while offsetting their individual weaknesses. Light microscopy has historically been a central method in correlative microscopy due to its widespread availability, compatibility with hydrated and live biological samples, and excellent molecular specificity through fluorescence labeling. However, conventional light microscopy can only achieve a resolution of ∼300 nm, undercutting its advantages in correlations with higher-resolution methods. The rise of super-resolution microscopy (SRM) over the past decade has drastically improved the resolution of light microscopy to ∼10 nm, thus creating exciting new opportunities and challenges for correlative microscopy. Here we review how these challenges are addressed to effectively correlate SRM with other microscopy techniques, including light microscopy, electron microscopy, cryomicroscopy, atomic force microscopy, and various forms of spectroscopy. Though we emphasize biological studies, we also discuss the application of correlative SRM to materials characterization and single-molecule reactions. Finally, we point out current limitations and discuss possible future improvements and advances. We thus demonstrate how a correlative approach adds new dimensions of information and provides new opportunities in the fast-growing field of SRM.

Advances in molecular labeling, high throughput imaging and machine intelligence portend powerful functional cellular biochemistry tools.

PubMed

Price, Jeffrey H; Goodacre, Angela; Hahn, Klaus; Hodgson, Louis; Hunter, Edward A; Krajewski, Stanislaw; Murphy, Robert F; Rabinovich, Andrew; Reed, John C; Heynen, Susanne

2002-01-01

Cellular behavior is complex. Successfully understanding systems at ever-increasing complexity is fundamental to advances in modern science and unraveling the functional details of cellular behavior is no exception. We present a collection of prospectives to provide a glimpse of the techniques that will aid in collecting, managing and utilizing information on complex cellular processes via molecular imaging tools. These include: 1) visualizing intracellular protein activity with fluorescent markers, 2) high throughput (and automated) imaging of multilabeled cells in statistically significant numbers, and 3) machine intelligence to analyze subcellular image localization and pattern. Although not addressed here, the importance of combining cell-image-based information with detailed molecular structure and ligand-receptor binding models cannot be overlooked. Advanced molecular imaging techniques have the potential to impact cellular diagnostics for cancer screening, clinical correlations of tissue molecular patterns for cancer biology, and cellular molecular interactions for accelerating drug discovery. The goal of finally understanding all cellular components and behaviors will be achieved by advances in both instrumentation engineering (software and hardware) and molecular biochemistry. Copyright 2002 Wiley-Liss, Inc.

Characterization of humic acids by two-dimensional correlation fluorescence spectroscopy

NASA Astrophysics Data System (ADS)

Nakashima, K.; Xing, Shaoyong; Gong, Yongkuan; Miyajima, Toru

2008-07-01

We have investigated interaction between humic acids and heavy metal ions by fluorescence spectroscopy. The humic acids examined are Aldrich humic acid (AHA) and Dando humic acid (DHA), and heavy metal ions are Cu 2+ and Pb 2+. The binding constants between the humic acids and the heavy metal ions are obtained by a conventional fluorescence quenching technique. The two prominent bands in the fluorescence spectra of the humic acids give different binding constants, implying that the two bands are originated from different fluorescent species in the matrices of the humic acids. This was confirmed by two-dimensional correlation analysis based on the quenching perturbation on the fluorescence spectra. Two prominent cross peaks corresponding to the two fluorescence bands are obtained in the asynchronous maps, indicating that the two fluorescence bands belong to different species. The order of the response of the two fluorescence bands to the quenching perturbation is also elucidated based on Noda's rule.

Heavy Atom Effect of Bromine Significantly Enhances Exciton Utilization of Delayed Fluorescence Luminogens.

PubMed

Gan, Shifeng; Hu, Shimin; Li, Xiang-Long; Zeng, Jiajie; Zhang, Dongdong; Huang, Tianyu; Luo, Wenwen; Zhao, Zujin; Duan, Lian; Su, Shi-Jian; Tang, Ben Zhong

2018-05-23

Raising triplet exciton utilization of pure organic luminescent materials is of significant importance for efficiency advancement of organic light-emitting diodes (OLEDs). Herein, by introducing bromine atom(s) onto a typical molecule (bis(carbazol-9-yl)-4,5-dicyanobenzene) with thermally activated delayed fluorescence, we demonstrate that the heavy atom effect of bromine can increase spin-orbit coupling and promote the reverse intersystem crossing, which endow the molecules with more distinct delayed fluorescence. In consequence, the triplet exciton utilization is improved greatly with the increase of bromine atoms, affording apparently advanced external quantum efficiencies of OLEDs. Utilizing the enhancement effect of bromine atoms on delayed fluorescence should be a simple and promising design concept for efficient organic luminogens with high exciton utilization.

Spatiotemporal image correlation spectroscopy (STICS) theory, verification, and application to protein velocity mapping in living CHO cells.

PubMed

Hebert, Benedict; Costantino, Santiago; Wiseman, Paul W

2005-05-01

We introduce a new extension of image correlation spectroscopy (ICS) and image cross-correlation spectroscopy (ICCS) that relies on complete analysis of both the temporal and spatial correlation lags for intensity fluctuations from a laser-scanning microscopy image series. This new approach allows measurement of both diffusion coefficients and velocity vectors (magnitude and direction) for fluorescently labeled membrane proteins in living cells through monitoring of the time evolution of the full space-time correlation function. By using filtering in Fourier space to remove frequencies associated with immobile components, we are able to measure the protein transport even in the presence of a large fraction (>90%) of immobile species. We present the background theory, computer simulations, and analysis of measurements on fluorescent microspheres to demonstrate proof of principle, capabilities, and limitations of the method. We demonstrate mapping of flow vectors for mixed samples containing fluorescent microspheres with different emission wavelengths using space time image cross-correlation. We also present results from two-photon laser-scanning microscopy studies of alpha-actinin/enhanced green fluorescent protein fusion constructs at the basal membrane of living CHO cells. Using space-time image correlation spectroscopy (STICS), we are able to measure protein fluxes with magnitudes of mum/min from retracting lamellar regions and protrusions for adherent cells. We also demonstrate the measurement of correlated directed flows (magnitudes of mum/min) and diffusion of interacting alpha5 integrin/enhanced cyan fluorescent protein and alpha-actinin/enhanced yellow fluorescent protein within living CHO cells. The STICS method permits us to generate complete transport maps of proteins within subregions of the basal membrane even if the protein concentration is too high to perform single particle tracking measurements.

Miniature probe for the delivery and monitoring of a photopolymerizable material

NASA Astrophysics Data System (ADS)

Schmocker, Andreas; Khoushabi, Azadeh; Schizas, Constantin; Bourban, Pierre-Etienne; Pioletti, Dominique P.; Moser, Christophe

2015-12-01

Photopolymerization is a common method to cure materials initially in a liquid state, such as dental implants or bone or tissue fillers. Recent advances in the development of biocompatible gel- and cement-systems open up an avenue for in situ photopolymerization. For minimally invasive surgery, such procedures require miniaturized surgical endoscopic probes to activate and control photopolymerization in situ. We present a miniaturized light probe in which a photoactive material can be (1) mixed, pressurized, and injected, (2) photopolymerized/photoactivated, and (3) monitored during the chemical reaction. The device is used to implant and cure poly(ethylene glycol) dimethacrylate-hydrogel-precursor in situ with ultraviolet A (UVA) light (365 nm) while the polymerization reaction is monitored in real time by collecting the fluorescence and Raman signals generated by the 532-nm excitation light source. Hydrogels could be delivered, photopolymerized, and monitored by the probe up to a curing depth of 4 cm. The size of the photopolymerized samples could be correlated to the fluorescent signal collected by the probe, and the reproducibility of the procedure could be demonstrated. The position of the probe tip inside a bovine caudal intervertebral disc could be estimated in vitro based on the collected fluorescence and Raman signal.

Nano-fEM: protein localization using photo-activated localization microscopy and electron microscopy.

PubMed

Watanabe, Shigeki; Richards, Jackson; Hollopeter, Gunther; Hobson, Robert J; Davis, Wayne M; Jorgensen, Erik M

2012-12-03

Mapping the distribution of proteins is essential for understanding the function of proteins in a cell. Fluorescence microscopy is extensively used for protein localization, but subcellular context is often absent in fluorescence images. Immuno-electron microscopy, on the other hand, can localize proteins, but the technique is limited by a lack of compatible antibodies, poor preservation of morphology and because most antigens are not exposed to the specimen surface. Correlative approaches can acquire the fluorescence image from a whole cell first, either from immuno-fluorescence or genetically tagged proteins. The sample is then fixed and embedded for electron microscopy, and the images are correlated (1-3). However, the low-resolution fluorescence image and the lack of fiducial markers preclude the precise localization of proteins. Alternatively, fluorescence imaging can be done after preserving the specimen in plastic. In this approach, the block is sectioned, and fluorescence images and electron micrographs of the same section are correlated (4-7). However, the diffraction limit of light in the correlated image obscures the locations of individual molecules, and the fluorescence often extends beyond the boundary of the cell. Nano-resolution fluorescence electron microscopy (nano-fEM) is designed to localize proteins at nano-scale by imaging the same sections using photo-activated localization microscopy (PALM) and electron microscopy. PALM overcomes the diffraction limit by imaging individual fluorescent proteins and subsequently mapping the centroid of each fluorescent spot (8-10). We outline the nano-fEM technique in five steps. First, the sample is fixed and embedded using conditions that preserve the fluorescence of tagged proteins. Second, the resin blocks are sectioned into ultrathin segments (70-80 nm) that are mounted on a cover glass. Third, fluorescence is imaged in these sections using the Zeiss PALM microscope. Fourth, electron dense structures are imaged in these same sections using a scanning electron microscope. Fifth, the fluorescence and electron micrographs are aligned using gold particles as fiducial markers. In summary, the subcellular localization of fluorescently tagged proteins can be determined at nanometer resolution in approximately one week.

Enzyme activity and AGE formation in a model of AST glycoxidation by D-fructose in vitro.

PubMed

Bousova, Iva; Vukasović, Danka; Juretić, Dubravka; Palicka, Vladimir; Drsata, Jaroslav

2005-03-01

Non-enzymatic glycation as the chain reaction between reducing sugars and free amino groups of proteins has been shown to correlate with physiological ageing and severity of diabetes. The process involves oxidative steps (glycoxidation). In this paper, the effect of D-fructose as a reactive sugar on aspartate aminotransferase (AST) as a model protein was monitored by measurements of the enzyme activity and formation of fluorescent advanced glycation end products (AGEs). Change in the AST activity was considered as a measure of the overall protein damage caused by glycation, and total AGEs and pentosidine represent, at least partly, the formation of glycoxidation products. Catalytic activity of AST in an incubation mixture containing D-fructose (50 mmol L(-1)), decreased compared to control values to 42% (p < 0.05) and to 11% (p < 0.05) on the 5th and on 21st day of incubation, respectively. In the presence of fructose, total fluorescent AGEs concentration was significantly higher since 5th day of incubation (110%, p < 0.05) and the fluorescent pentosidine concentration from 15th day of incubation (117%, p < 0.05) compared to control values, respectively. Catalytic activity of AST clearly and quantitatively demonstrated functional changes in the enzyme molecule caused by structural modifications initiated by fructose, while the evaluation of AGE formation and especially that of pentosidine by fluorescence measurement was less reliable.

Uptake and localisation of mTHPC (Foscan®) and its14C-labelled form in normal and tumour tissues of the hamster squamous cell carcinoma model: a comparative study

PubMed Central

Blant, S Andrejevic; Glanzmann, T M; Ballini, J-P; Wagnières, G; van den Bergh, H; Monnier, P

2002-01-01

The aim of this study was to evaluate the pharmacokinetics of meta(tetrahydroxyphenyl)chlorin (mTHPC) on different tissues of interest in a hamster tumour model and to confirm our earlier animal studies on semi-quantitative fluorescence microscopy. The results obtained by three different evaluation methods were compared: in vivo spectrofluorometry, ex vivo fluorescence microscopy and chemical extraction of 14C-labelled mTHPC. Following intracardiac injection of 0.5 mg kg−1 mTHPC, groups of five tumour-bearing animals were used for in situ light-induced fluorescence spectroscopy. Afterwards, the biopsies were taken and snap frozen for fluorescence microscopy. The presence of radioactivity in serum and tissues was determined after chemical digestion in scintillation fluid using a scintillation counter. For each analysed tissue, a good correlation was observed between the three evaluation methods. The highest fluorescence intensity and quantities of mTHPC were observed between 12 and 24 h in liver, kidney, serum, vascular endothelium and advanced neoplasia. The majority of mTHPC was found at around 48 h in smooth muscle and at 96 h in healthy cheek pouch mucosa and early malignant lesions. The lowest level of mTHPC was noted in striated muscle at all times. No selectivity in dye localisation was observed between early squamous cell carcinoma and healthy mucosa. Soon after the injection, a significant selectivity was noted for advanced squamous cell carcinoma as compared to healthy cheek pouch mucosa or striated muscle. A significant difference in mTHPC localisation and quantity was also observed between striated and smooth muscle during the first 48 h following the injection. Finally, this study demonstrated the usefulness of non-invasive in situ spectroscopic measurements to be performed systematically prior to photodynamic therapy as a real-time monitoring for each treated patient in order to individualise and adapt the light dosimetry and avoid over or under treatments. British Journal of Cancer (2002) 87, 1470–1478. doi:10.1038/sj.bjc.6600651 www.bjcancer.com © 2002 Cancer Research UK PMID:12454779

Spatial fluorescence cross-correlation spectroscopy between core and ring pinholes

NASA Astrophysics Data System (ADS)

Blancquaert, Yoann; Delon, Antoine; Derouard, Jacques; Jaffiol, Rodolphe

2006-04-01

Fluorescence Correlation Spectroscopy (FCS) is an attractive method to measure molecular concentration, mobility parameters and chemical kinetics. However its ability to descriminate different diffusing species needs to be improved. Recently, we have proposed a simplified spatial Fluorescence cross Correlation Spectroscopy (sFCCS) method, allowing, with only one focused laser beam to obtain two confocal volumes spatially shifted. Now, we present a new sFCCS optical geometry where the two pinholes, a ring and core, are encapsulated one in the other. In this approach all physical and chemical processes that occur in a single volume, like singlet-triplet dynamics and photobleaching, can be eliminated; moreover, this new optical geometry optimises the collection of fluorescence. The first cross Correlation curves for Rhodamine 6G (Rh6G) in Ethanol are presented, in addition to the effect of the size of fluorescent particules (nano-beads, diameters : 20, 100 and 200 nm). The relative simplicity of the method leads us to propose sFCCS as an appropriate method for the determination of diffusion parameters of fluorophores in solution or cells. Nevertheless, progresses in the ingeniering of the optical Molecular Detection Efficiency volumes are highly desirable, in order to improve the descrimination between the cross correlated volumes.

A trifurcated fiber-optic-probe-based optical system designed for AGEs measurement

NASA Astrophysics Data System (ADS)

Wang, Yikun; Zhang, Long; Zhu, Ling; Liu, Yong; Zhang, Gong; Wang, An

2012-03-01

Advanced Glycation End-products (AGEs) are biochemical end-products of non-enzymatic glycation and are formed irreversibly in human serum and skin tissue. AGEs are thought to play an important role in the pathogenesis of diabetes and corresponding complications. All conventional methods for measuring AGEs must take sampling and measure in vitro. These methods are invasive and have the problem of relatively time-consuming. AGEs have fluorescent characteristics. Skin AGEs can be assessed noninvasively by collecting the fluorescence emitted from skin tissue when excited with proper light. However, skin tissue has absorption and scattering effects on fluorescence of AGEs, it is not reliable to evaluate the accumulation of AGEs according the emitted fluorescence but not considering optical properties of skin tissue. In this study, a portable system for detecting AGEs fluorescence and skin reflectance spectrum simultaneously has been developed. The system mainly consists of an ultraviolet light source, a broadband light source, a trifurcated fiber-optic probe, and a compact charge coupled device (CCD) spectrometer. The fiber-optic probe consists of 36 optical fibers which are connected to the ultraviolet light source, 6 optical fibers connected to the broadband light source, and a core fiber connected to the CCD spectrometer. Demonstrative test measurements with the system on skin tissue of 40 healthy subjects have been performed. Using parameters that are calculated from skin reflectance spectrum, the distortion effects caused by skin absorption and scattering can be eliminated, and the integral intensity of corrected fluorescence has a strong correlation with the accumulation of AGEs. The system looks very promising for both laboratory and clinical applications to monitor AGEs related diseases, especially for chronic diabetes and complications.

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.

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.

Live-cell imaging of cell signaling using genetically encoded fluorescent reporters.

PubMed

Ni, Qiang; Mehta, Sohum; Zhang, Jin

2018-01-01

Synergistic advances in fluorescent protein engineering and live-cell imaging techniques in recent years have fueled the concurrent development and application of genetically encoded fluorescent reporters that are tailored for tracking signaling dynamics in living systems over multiple length and time scales. These biosensors are uniquely suited for this challenging task, owing to their specificity, sensitivity, and versatility, as well as to the noninvasive and nondestructive nature of fluorescence and the power of genetic encoding. Over the past 10 years, a growing number of fluorescent reporters have been developed for tracking a wide range of biological signals in living cells and animals, including second messenger and metabolite dynamics, enzyme activation and activity, and cell cycle progression and neuronal activity. Many of these biosensors are gaining wide use and are proving to be indispensable for unraveling the complex biological functions of individual signaling molecules in their native environment, the living cell, shedding new light on the structural and molecular underpinnings of cell signaling. In this review, we highlight recent advances in protein engineering that are likely to help expand and improve the design and application of these valuable tools. We then turn our focus to specific examples of live-cell imaging using genetically encoded fluorescent reporters as an important platform for advancing our understanding of G protein-coupled receptor signaling and neuronal activity. © 2017 Federation of European Biochemical Societies.

Fluorescence decay time imaging using an imaging photon detector with a radio frequency photon correlation system

NASA Astrophysics Data System (ADS)

Morgan, Christopher G.; Mitchell, A. C.; Murray, J. G.

1990-05-01

An imaging photon detector has been modified to incorporate fast timing electronics coupled to a custom built photon correlator interfaced to a RISC computer. Using excitation with intensity- muodulated light, fluorescence images can be readily obtained where contrast is determined by the decay time of emission, rather than by intensity. This technology is readily extended to multifrequency phase/demodulation fluorescence imaging or to differential polarised phase fluorometry. The potential use of the correlator for confocal imaging with a laser scanner is also briefly discussed.

In situ accumulation of advanced glycation endproducts (AGEs) in bone matrix and its correlation with osteoclastic bone resorption.

PubMed

Dong, X Neil; Qin, An; Xu, Jiake; Wang, Xiaodu

2011-08-01

Advanced glycation end products (AGEs) have been observed to accumulate in bone with increasing age and may impose effects on bone resorption activities. However, the underlying mechanism of AGEs accumulation in bone is still poorly understood. In this study, human cortical bone specimens from young (31±6years old), middle-aged (51±3years old) and elderly (76±4years old) groups were examined to determine the spatial-temporal distribution of AGEs in bone matrix and its effect on bone resorption activities by directly culturing osteoclastic cells on bone slices. The results of this study indicated that the fluorescence intensity (excitation wave length 360nm and emission wave length 470±40nm) could be used to estimate the relative distribution of AGEs in bone (pentosidine as its marker) under an epifluorescence microscope. Using the fluorescence intensity as the relative measure of AGEs concentration, it was found that the concentration of AGEs varied with biological tissue ages, showing the greatest amount in the interstitial tissue, followed by the old osteons, and the least amount in newly formed osteons. In addition, AGEs accumulation was found to be dependent on donor ages, suggesting that the younger the donor the less AGEs were accumulated in the tissue. Most interestingly, AGEs accumulation appeared to initiate from the region of cement lines, and spread diffusively to the other parts as the tissue aged. Finally, it was observed that the bone resorption activities of osteoclasts were positively correlated with the in situ concentration of AGEs and such an effect was enhanced with increasing donor age. These findings may help elucidate the mechanism of AGEs accumulation in bone and its association with bone remodeling process. Copyright © 2011 Elsevier Inc. All rights reserved.

IN SITU ACCUMULATION OF ADVANCED GLYCATION ENDPRODUCTS (AGES) IN BONE MATRIX AND ITS CORRELATION WITH OSTEOCLASTIC BONE RESORPTION

PubMed Central

Dong, X. Neil; Qin, An; Xu, Jiake; Wang, Xiaodu

2011-01-01

Advanced glycation end products (AGEs) have been observed to accumulate in bone with increasing age and may impose effects on bone resorption activities. However, the underlying mechanism of AGEs accumulation in bone is still poorly understood. In this study, human cortical bone specimens from young (31±6 years old), middle-aged (51±3 years old) and elderly (76±4 years old) groups were examined to determine the spatial-temporal distribution of AGEs in bone matrix and its effect on bone resorption activities by directly culturing osteoclastic cells on bone slices. The results of this study indicated that the fluorescence intensity (excitation wave length 360 nm and emission wave length 470±40 nm) could be used to estimate the relative distribution of AGEs in bone (pentosidine as its marker) under an epifluorescence microscope. Using the fluorescence intensity as the relative measure of AGEs concentration, it was found that the concentration of AGEs varied with biological tissue ages, showing the greatest amount in the interstitial tissue, followed by the old osteons, and the least amount in newly formed osteons. In addition, AGEs accumulation was found to be dependent on donor ages, suggesting that the younger the donor the less AGEs were accumulated in the tissue. Most interestingly, AGEs accumulation appeared to initiate from the region of cement lines, and spread diffusively to the other parts as the tissue aged. Finally, it was observed that the bone resorption activities of osteoclasts were positively correlated with the in situ concentration of AGEs and such an effect was enhanced with increasing donor age. These findings may help elucidate the mechanism of AGEs accumulation in bone and its association with bone remodeling process. PMID:21530698

In Vivo Fluorescence Correlation and Cross-Correlation Spectroscopy

NASA Astrophysics Data System (ADS)

Mütze, Jörg; Ohrt, Thomas; Petrášek, Zdeněk; Schwille, Petra

In this manuscript, we describe the application of Fluorescence Correlation Spectroscopy (FCS), Fluorescence Cross-Correlation Spectroscopy (FCCS), and scanning FCS (sFCS) to two in vivo systems. In the first part, we describe the application of two-photon standard and scanning FCS in Caenorhabditis elegans embryos. The differentiation of a single fertilized egg into a complex organism in C. elegans is regulated by a number of protein-dependent processes. The oocyte divides asymmetrically into two daughter cells of different developmental fate. Two of the involved proteins, PAR-2 and NMY-2, are studied. The second investigated system is the mechanism of RNA interference in human cells. An EGFP based cell line that allows to study the dynamics and localization of the RNA-induced silencing complex (RISC) with FCS in vivo is created, which has so far been inaccessible with other experimental methods. Furthermore, Fluorescence Cross-Correlation Spectroscopy is employed to highlight the asymmetric incorporation of labeled siRNAs into RISC.

Simultaneous Correlative Scanning Electron and High-NA Fluorescence Microscopy

PubMed Central

Liv, Nalan; Zonnevylle, A. Christiaan; Narvaez, Angela C.; Effting, Andries P. J.; Voorneveld, Philip W.; Lucas, Miriam S.; Hardwick, James C.; Wepf, Roger A.; Kruit, Pieter; Hoogenboom, Jacob P.

2013-01-01

Correlative light and electron microscopy (CLEM) is a unique method for investigating biological structure-function relations. With CLEM protein distributions visualized in fluorescence can be mapped onto the cellular ultrastructure measured with electron microscopy. Widespread application of correlative microscopy is hampered by elaborate experimental procedures related foremost to retrieving regions of interest in both modalities and/or compromises in integrated approaches. We present a novel approach to correlative microscopy, in which a high numerical aperture epi-fluorescence microscope and a scanning electron microscope illuminate the same area of a sample at the same time. This removes the need for retrieval of regions of interest leading to a drastic reduction of inspection times and the possibility for quantitative investigations of large areas and datasets with correlative microscopy. We demonstrate Simultaneous CLEM (SCLEM) analyzing cell-cell connections and membrane protrusions in whole uncoated colon adenocarcinoma cell line cells stained for actin and cortactin with AlexaFluor488. SCLEM imaging of coverglass-mounted tissue sections with both electron-dense and fluorescence staining is also shown. PMID:23409024

Two-dimensional fluorescence correlation spectroscopy: resolution of fluorescence of tryptophan residues in horse heart myoglobin.

PubMed

Nakashima, Kenichi; Yuda, Kazuki; Ozaki, Yukihiro; Noda, Isao

2003-11-01

Generalized two-dimensional (2D) fluorescence correlation spectroscopy has been used to resolve fluorescence of two tryptophan (Trp) residues in horse heart myoglobin. Fluorescence quenching is employed as a perturbation mode for causing intensity changes in the fluorescence (quenching perturbation). Two kinds of quenchers, iodide ion and acrylamide, are used for inducing fluorescence intensity change. This technique works because the Trp residue located at the 7th position (W7) is known to be easily accessible to the quencher, whereas that located at the 14th position (W14) is not. By this technique, the fluorescence spectra of the two Trp residues were clearly resolved. From asynchronous maps, it was also shown that the quenching of W7 fluorescence is brought about prior to the quenching of W14 fluorescence. This result is consistent with the structure of horse heart myoglobin that was proposed earlier. Furthermore, it was elucidated that the present 2D analysis is not interfered with by Raman bands of the solvents, which sometimes brings difficulty into conventional fluorescence analysis.

Fluorescence correlation spectroscopy: novel variations of an established technique.

PubMed

Haustein, Elke; Schwille, Petra

2007-01-01

Fluorescence correlation spectroscopy (FCS) is one of the major biophysical techniques used for unraveling molecular interactions in vitro and in vivo. It allows minimally invasive study of dynamic processes in biological specimens with extremely high temporal and spatial resolution. By recording and correlating the fluorescence fluctuations of single labeled molecules through the exciting laser beam, FCS gives information on molecular mobility and photophysical and photochemical reactions. By using dual-color fluorescence cross-correlation, highly specific binding studies can be performed. These have been extended to four reaction partners accessible by multicolor applications. Alternative detection schemes shift accessible time frames to slower processes (e.g., scanning FCS) or higher concentrations (e.g., TIR-FCS). Despite its long tradition, FCS is by no means dated. Rather, it has proven to be a highly versatile technique that can easily be adapted to solve specific biological questions, and it continues to find exciting applications in biology and medicine.

Correlative fluorescence microscopy and scanning transmission electron microscopy of quantum-dot-labeled proteins in whole cells in liquid.

PubMed

Dukes, Madeline J; Peckys, Diana B; de Jonge, Niels

2010-07-27

Correlative fluorescence microscopy and transmission electron microscopy (TEM) is a state-of-the-art microscopy methodology to study cellular function, combining the functionality of light microscopy with the high resolution of electron microscopy. However, this technique involves complex sample preparation procedures due to its need for either thin sections or frozen samples for TEM imaging. Here, we introduce a novel correlative approach capable of imaging whole eukaryotic cells in liquid with fluorescence microscopy and with scanning transmission electron microscopy (STEM); there is no additional sample preparation necessary for the electron microscopy. Quantum dots (QDs) were bound to epidermal growth factor (EGF) receptors of COS7 fibroblast cells. Fixed whole cells in saline water were imaged with fluorescence microscopy and subsequently with STEM. The STEM images were correlated with fluorescence images of the same cellular regions. QDs of dimensions 7x12 nm were visible in a 5 microm thick layer of saline water, consistent with calculations. A spatial resolution of 3 nm was achieved on the QDs.

Correlative Fluorescence Microscopy and Scanning Transmission Electron Microscopy of Quantum Dot Labeled Proteins in Whole Cells in Liquid

PubMed Central

Dukes, Madeline J.; Peckys, Diana B.; de Jonge, Niels

2010-01-01

Correlative fluorescence microscopy and transmission electron microscopy (TEM) is a state-of-the-art microscopy methodology to study cellular function, combining the functionality of light microscopy with the high resolution of electron microscopy. However, this technique involves complex sample preparation procedures due to its need for either thin sections or frozen samples for TEM imaging. Here, we introduce a novel correlative approach capable of imaging whole eukaryotic cells in liquid with fluorescence microscopy and with scanning transmission electron microscopy (STEM); there is no additional sample preparation necessary for the electron microscopy. Quantum dots (QDs) were bound to epidermal growth factor (EGF) receptors of COS7 fibroblast cells. Fixed whole cells in saline water were imaged with fluorescence microscopy and subsequently with STEM. The STEM images were correlated with fluorescence images of the same cellular regions. QDs of dimensions 7 × 12 nm were visible in a 5 μm thick layer of saline water, consistent with calculations. A spatial resolution of 3 nm was achieved on the QDs. PMID:20550177

Near-Infrared Fluorescence-Enhanced Optical Tomography

PubMed Central

2016-01-01

Fluorescence-enhanced optical imaging using near-infrared (NIR) light developed for in vivo molecular targeting and reporting of cancer provides promising opportunities for diagnostic imaging. The current state of the art of NIR fluorescence-enhanced optical tomography is reviewed in the context of the principle of fluorescence, the different measurement schemes employed, and the mathematical tools established to tomographically reconstruct the fluorescence optical properties in various tissue domains. Finally, we discuss the recent advances in forward modeling and distributed memory parallel computation to provide robust, accurate, and fast fluorescence-enhanced optical tomography. PMID:27803924

Near-Infrared Fluorescence-Enhanced Optical Tomography.

PubMed

Zhu, Banghe; Godavarty, Anuradha

2016-01-01

Fluorescence-enhanced optical imaging using near-infrared (NIR) light developed for in vivo molecular targeting and reporting of cancer provides promising opportunities for diagnostic imaging. The current state of the art of NIR fluorescence-enhanced optical tomography is reviewed in the context of the principle of fluorescence, the different measurement schemes employed, and the mathematical tools established to tomographically reconstruct the fluorescence optical properties in various tissue domains. Finally, we discuss the recent advances in forward modeling and distributed memory parallel computation to provide robust, accurate, and fast fluorescence-enhanced optical tomography.

Experimental design and quality assurance: in situ fluorescence instrumentation

USGS Publications Warehouse

Conmy, Robyn N.; Del Castillo, Carlos E.; Downing, Bryan D.; Chen, Robert F.

2014-01-01

Both instrument design and capabilities of fluorescence spectroscopy have greatly advanced over the last several decades. Advancements include solid-state excitation sources, integration of fiber optic technology, highly sensitive multichannel detectors, rapid-scan monochromators, sensitive spectral correction techniques, and improve data manipulation software (Christian et al., 1981, Lochmuller and Saavedra, 1986; Cabniss and Shuman, 1987; Lakowicz, 2006; Hudson et al., 2007). The cumulative effect of these improvements have pushed the limits and expanded the application of fluorescence techniques to numerous scientific research fields. One of the more powerful advancements is the ability to obtain in situ fluorescence measurements of natural waters (Moore, 1994). The development of submersible fluorescence instruments has been made possible by component miniaturization and power reduction including advances in light sources technologies (light-emitting diodes, xenon lamps, ultraviolet [UV] lasers) and the compatible integration of new optical instruments with various sampling platforms (Twardowski et at., 2005 and references therein). The development of robust field sensors skirt the need for cumbersome and or time-consuming filtration techniques, the potential artifacts associated with sample storage, and coarse sampling designs by increasing spatiotemporal resolution (Chen, 1999; Robinson and Glenn, 1999). The ability to obtain rapid, high-quality, highly sensitive measurements over steep gradients has revolutionized investigations of dissolved organic matter (DOM) optical properties, thereby enabling researchers to address novel biogeochemical questions regarding colored or chromophoric DOM (CDOM). This chapter is dedicated to the origin, design, calibration, and use of in situ field fluorometers. It will serve as a review of considerations to be accounted for during the operation of fluorescence field sensors and call attention to areas of concern when making this type of measurement. Attention is also given to ways in which in-water fluorescence measurements have revolutionized biogeochemical studies of CDOM and how those measurements can be used in conjunction with remotely sense satellite data to understand better the biogeochemistry of DOM in aquatic environments.

SOPROCARE - 450 nm wavelength detection tool for microbial plaque and gingival inflammation: a clinical study

NASA Astrophysics Data System (ADS)

Rechmann, P.; Liou, Shasan W.; Rechmann, Beate M.; Featherstone, John D.

2014-02-01

Gingivitis due to microbial plaque and calculus can lead over time if left untreated to advanced periodontal disease with non-physiological pocket formation. Removal of microbial plaque in the gingivitis stage typically achieves gingival health. The SOPROCARE camera system emits blue light at 450 nm wavelength using three blue diodes. The 450 nm wavelength is located in the non-ionizing, visible spectral wavelength region and thus is not dangerous. It is assumed that using the SOPROCARE camera in perio-mode inflamed gingiva can easily be observed and inflammation can be scored due to fluorescence from porphyrins in blood. The assumption is also that illumination of microbial plaque with blue light induces fluorescence due to the bacteria and porphyrin content of the plaque and thus can help to make microbial plaque and calculus visible. Aim of the study with 55 subjects was to evaluate the ability of the SOPROCARE fluorescence camera system to detect, visualize and allow scoring of microbial plaque in comparison to the Turesky modification of the Quigley and Hein plaque index. A second goal was to detect and score gingival inflammation and correlated the findings to the Silness and Löe gingival inflammation index. The study showed that scoring of microbial plaque as well as gingival inflammation levels similar to the established Turesky modified Quigley Hein index and the Silness and Löe gingival inflammation index can easily be done using the SOPROCARE fluorescence system in periomode. Linear regression fits between the different clinical indices and SOPROCARE scores in fluorescence perio-mode revealed the system's capacity for effective discrimination between scores.

Development of a homogeneous assay format for p53 antibodies using fluorescence correlation spectroscopy

NASA Astrophysics Data System (ADS)

Neuweiler, Hannes; Scheffler, Silvia; Sauer, Markus

2005-08-01

The development of reliable methods for the detection of minute amounts of antibodies directly in homogeneous solution represents one of the major tasks in the current research field of molecular diagnostics. We demonstrate the potential of fluorescence correlation spectroscopy (FCS) in combination with quenched peptide-based fluorescence probes for sensitive detection of p53 antibodies directly in homogeneous solution. Single tryptophan (Trp) residues in the sequences of short, synthetic peptide epitopes of the human p53 protein efficiently quench the fluorescence of an oxazine fluorophore attached to the amino terminal ends of the peptides. The fluorescence quenching mechanism is thought to be a photoinduced electron transfer reaction from Trp to the dye enabled by the formation of intramolecular complexes between dye and Trp. Specific recognition of the epitope by the antibody confines the conformational flexibility of the peptide. Consequently, complex formation between dye and Trp is abolished and fluorescence is recovered. Using fluorescence correlation spectroscopy (FCS), antibody binding can be monitored observing two parameters simultaneously: the diffusional mobility of the peptide as well as the quenching amplitude induced by the conformational flexibility of the peptide change significantly upon antibody binding. Our data demonstrate that FCS in combination with fluorescence-quenched peptide epitopes opens new possibilities for the reliable detection of antibody binding events in homogeneous solution.

Integrated circuit-based electrochemical sensor for spatially resolved detection of redox-active metabolites in biofilms.

PubMed

Bellin, Daniel L; Sakhtah, Hassan; Rosenstein, Jacob K; Levine, Peter M; Thimot, Jordan; Emmett, Kevin; Dietrich, Lars E P; Shepard, Kenneth L

2014-01-01

Despite advances in monitoring spatiotemporal expression patterns of genes and proteins with fluorescent probes, direct detection of metabolites and small molecules remains challenging. A technique for spatially resolved detection of small molecules would benefit the study of redox-active metabolites that are produced by microbial biofilms and can affect their development. Here we present an integrated circuit-based electrochemical sensing platform featuring an array of working electrodes and parallel potentiostat channels. 'Images' over a 3.25 × 0.9 mm(2) area can be captured with a diffusion-limited spatial resolution of 750 μm. We demonstrate that square wave voltammetry can be used to detect, identify and quantify (for concentrations as low as 2.6 μM) four distinct redox-active metabolites called phenazines. We characterize phenazine production in both wild-type and mutant Pseudomonas aeruginosa PA14 colony biofilms, and find correlations with fluorescent reporter imaging of phenazine biosynthetic gene expression.

Integrated circuit-based electrochemical sensor for spatially resolved detection of redox-active metabolites in biofilms

PubMed Central

Bellin, Daniel L.; Sakhtah, Hassan; Rosenstein, Jacob K.; Levine, Peter M.; Thimot, Jordan; Emmett, Kevin; Dietrich, Lars E. P.; Shepard, Kenneth L.

2014-01-01

Despite advances in monitoring spatiotemporal expression patterns of genes and proteins with fluorescent probes, direct detection of metabolites and small molecules remains challenging. A technique for spatially resolved detection of small molecules would benefit the study of redox-active metabolites produced by microbial biofilms, which can drastically affect colony development. Here we present an integrated circuit-based electrochemical sensing platform featuring an array of working electrodes and parallel potentiostat channels. “Images” over a 3.25 × 0.9 mm area can be captured with a diffusion-limited spatial resolution of 750 μm. We demonstrate that square wave voltammetry can be used to detect, identify, and quantify (for concentrations as low as 2.6 μM) four distinct redox-active metabolites called phenazines. We characterize phenazine production in both wild-type and mutant Pseudomonas aeruginosa PA14 colony biofilms, and find correlations with fluorescent reporter imaging of phenazine biosynthetic gene expression. PMID:24510163

[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.

Variations in the endogenous fluorescence of rabbit corneas after mechanical property alterations

NASA Astrophysics Data System (ADS)

Ortega-Martinez, Antonio; Touchette, Genna; Zhu, Hong; Kochevar, Irene E.; Franco, Walfre

2017-09-01

Keratoconus is an eye disease in which the cornea progressively deforms due to loss of cornea mechanical rigidity, and thus causes deterioration of visual acuity. Techniques to characterize the mechanical characteristics of the cornea are important to better monitor changes and response to treatments. To investigate the feasibility of using the endogenous fluorescence of cornea for monitoring alterations of its mechanical rigidity, linear tensiometry was used to quantitate stiffness and Young's modulus (YM) after treatments that increase cornea stiffness (collagen photocross-linking) or decrease stiffness (enzymatic digestion). The endogenous ultraviolet fluorescence of cornea was also measured before and after these treatments. The fluorescence excitation/emission spectral ranges were 280 to 430/390 to 520 nm, respectively. A correlation analysis was carried out to identify fluorescence excitation/emission pairs whose intensity changes correlated with the stiffness. A positive correlation was found between variations in fluorescence intensity of the 415-/485-nm excitation/emission pair and YM of photocross-linked corneas. After treatment of corneas with pepsin, the YM decreased as the fluorescence intensity at 290-/390-nm wavelengths decreased. For weakening of corneas with collagenase, only qualitative changes in the fluorescence spectrum were observed. Changes in the concentration of native or newly created fluorescent molecular species contain information that may be directly or indirectly related to the mechanical structure of the cornea.

Next Generation of Advanced Laser Fluorescence Technology for Characterization of Natural Aquatic Environments

DTIC Science & Technology

2011-09-01

project research addresses our long-term goal to develop an analytical suite of the Advanced Laser Fluorescence (ALF) methods and instruments to improve...demonstrated ALF utility as an integrated tool for aquatic research and observations. The ALF integration into the major oceanographic programs is...currently in progress, including the California Current Ecosystem Long Term Ecological Research (CCE LTER, NSF) and California Cooperative Oceanic

Dual PET and Near-Infrared Fluorescence Imaging Probes as Tools for Imaging in Oncology

PubMed Central

An, Fei-Fei; Chan, Mark; Kommidi, Harikrishna; Ting, Richard

2016-01-01

OBJECTIVE The purpose of this article is to summarize advances in PET fluorescence resolution, agent design, and preclinical imaging that make a growing case for clinical PET fluorescence imaging. CONCLUSION Existing SPECT, PET, fluorescence, and MRI contrast imaging techniques are already deeply integrated into the management of cancer, from initial diagnosis to the observation and management of metastases. Combined positron-emitting fluorescent contrast agents can convey new or substantial benefits that improve on these proven clinical contrast agents. PMID:27223168

Near-infrared laser-induced fluorescence detection in capillary electrophoresis.

PubMed

McWhorter, S; Soper, S A

2000-04-01

As capillary electrophoresis continues to focus on miniaturization, either through reducing column dimensions or situating entire electrophoresis systems on planar chips, advances in detection become necessary to meet the challenges posed by these electrophoresis platforms. The challenges result from the fact that miniaturization requires smaller load volumes, demanding highly sensitive detection. In addition, many times multiple targets must be analyzed simultaneously (multiplexed applications), further complicating detection. Near-infrared (NIR) fluorescence offers an attractive alternative to visible fluorescence for critical applications in capillary electrophoresis due to the impressive limits of detection that can be generated, in part resulting from the low background levels that are observed in the NIR. Advances in instrumentation and fluorogenic labels appropriate for NIR monitoring have led to a growing number of examples of the use of NIR fluorescence in capillary electrophoresis. In this review, we will cover instrumental components used to construct ultrasensitive NIR fluorescence detectors, including light sources and photon transducers. In addition, we will discuss various types of labeling dyes appropriate for NIR fluorescence and finally, we will present several applications that have used NIR fluorescence in capillary electrophoresis, especially for DNA sequencing and fragment analysis.

Design of multifunctional nanoparticles for combined in-vivo imaging and advanced drug delivery

NASA Astrophysics Data System (ADS)

Leary, James F.

2018-02-01

Design of multifunctional nanoparticles for multimodal in-vivo imaging and advanced targeting to diseased single cells for massive parallel processing nanomedicine approaches requires careful overall design and a multilayered approach. Initial core materials can include non-toxic metals which not only serve as an x-ray contrast agent for CAT scan imaging, but can contain T1 or T2 contrast agents for MRI imaging. One choice is superparamagnetic iron oxide NPs which also allow for convenient magnetic manipulation during manufacturing but also for re-positioning inside the body and for single cell hyperthermia therapies. To permit real-time fluorescence-guided surgery, fluorescence molecules can be included. Advanced targeting can be achieved by attaching antibodies, peptides, aptamers, or other targeting molecules to the nanoparticle in a multilayered approach producing "programmable nanoparticles" whereby the "programming" means controlling a sequence of multi-step targeting methods. Addition of membrane permeating peptides can facilitate uptake by the cell. Addition of "stealth" molecules (e.g. PEG or chitosan) to the outer surfaces of the nanoparticles can permit greatly enhanced circulation times in-vivo which in turn lead to lower amounts of drug exposure to the patient which can reduce undesirable side effects. Nanoparticles with incomplete layers can be removed by affinity purification methods to minimize mistargeting events in-vivo. Nanoscale imaging of these manufactured, multifunctional nanoparticles can be achieved either directly through superresolution microscopy or indirectly through single nanoparticle zeta-sizing or x-ray correlation microscopy. Since these multifunctional nanoparticles are best analyzed by technologies permitting analysis in aqueous environments, superresolution microscopy is, in most cases, the preferred method.

Dendrimer probes for enhanced photostability and localization in fluorescence imaging.

PubMed

Kim, Younghoon; Kim, Sung Hoon; Tanyeri, Melikhan; Katzenellenbogen, John A; Schroeder, Charles M

2013-04-02

Recent advances in fluorescence microscopy have enabled high-resolution imaging and tracking of single proteins and biomolecules in cells. To achieve high spatial resolutions in the nanometer range, bright and photostable fluorescent probes are critically required. From this view, there is a strong need for development of advanced fluorescent probes with molecular-scale dimensions for fluorescence imaging. Polymer-based dendrimer nanoconjugates hold strong potential to serve as versatile fluorescent probes due to an intrinsic capacity for tailored spectral properties such as brightness and emission wavelength. In this work, we report a new, to our knowledge, class of molecular probes based on dye-conjugated dendrimers for fluorescence imaging and single-molecule fluorescence microscopy. We engineered fluorescent dendritic nanoprobes (FDNs) to contain multiple organic dyes and reactive groups for target-specific biomolecule labeling. The photophysical properties of dye-conjugated FDNs (Cy5-FDNs and Cy3-FDNs) were characterized using single-molecule fluorescence microscopy, which revealed greatly enhanced photostability, increased probe brightness, and improved localization precision in high-resolution fluorescence imaging compared to single organic dyes. As proof-of-principle demonstration, Cy5-FDNs were used to assay single-molecule nucleic acid hybridization and for immunofluorescence imaging of microtubules in cytoskeletal networks. In addition, Cy5-FDNs were used as reporter probes in a single-molecule protein pull-down assay to characterize antibody binding and target protein capture. In all cases, the photophysical properties of FDNs resulted in enhanced fluorescence imaging via improved brightness and/or photostability. Copyright © 2013 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Resolving colocalization of bacteria and metal(loid)s on plant root surfaces by combining fluorescence in situ hybridization (FISH) with multiple-energy micro-focused X-ray fluorescence (ME μXRF).

PubMed

Honeker, Linnea K; Root, Robert A; Chorover, Jon; Maier, Raina M

2016-12-01

Metal(loid)-contamination of the environment due to anthropogenic activities is a global problem. Understanding the fate of contaminants requires elucidation of biotic and abiotic factors that influence metal(loid) speciation from molecular to field scales. Improved methods are needed to assess micro-scale processes, such as those occurring at biogeochemical interfaces between plant tissues, microbial cells, and metal(loid)s. Here we present an advanced method that combines fluorescence in situ hybridization (FISH) with synchrotron-based multiple-energy micro-focused X-ray fluorescence microprobe imaging (ME μXRF) to examine colocalization of bacteria and metal(loid)s on root surfaces of plants used to phytostabilize metalliferous mine tailings. Bacteria were visualized on a small root section using SytoBC nucleic acid stain and FISH probes targeting the domain Bacteria and a specific group (Alphaproteobacteria, Gammaproteobacteria, or Actinobacteria). The same root region was then analyzed for elemental distribution and metal(loid) speciation of As and Fe using ME μXRF. The FISH and ME μXRF images were aligned using ImageJ software to correlate microbiological and geochemical results. Results from quantitative analysis of colocalization show a significantly higher fraction of As colocalized with Fe-oxide plaques on the root surfaces (fraction of overlap 0.49±0.19) than to bacteria (0.072±0.052) (p<0.05). Of the bacteria that colocalized with metal(loid)s, Actinobacteria, known for their metal tolerance, had a higher correlation with both As and Fe than Alphaproteobacteria or Gammaproteobacteria. This method demonstrates how coupling these micro-techniques can expand our understanding of micro-scale interactions between roots, metal(loid)s and microbes, information that should lead to improved mechanistic models of metal(loid) speciation and fate. Copyright © 2016 Elsevier B.V. All rights reserved.

A 32-channel photon counting module with embedded auto/cross-correlators for real-time parallel fluorescence correlation spectroscopy

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

Gong, S.; Labanca, I.; Rech, I.

2014-10-15

Fluorescence correlation spectroscopy (FCS) is a well-established technique to study binding interactions or the diffusion of fluorescently labeled biomolecules in vitro and in vivo. Fast FCS experiments require parallel data acquisition and analysis which can be achieved by exploiting a multi-channel Single Photon Avalanche Diode (SPAD) array and a corresponding multi-input correlator. This paper reports a 32-channel FPGA based correlator able to perform 32 auto/cross-correlations simultaneously over a lag-time ranging from 10 ns up to 150 ms. The correlator is included in a 32 × 1 SPAD array module, providing a compact and flexible instrument for high throughput FCS experiments.more » However, some inherent features of SPAD arrays, namely afterpulsing and optical crosstalk effects, may introduce distortions in the measurement of auto- and cross-correlation functions. We investigated these limitations to assess their impact on the module and evaluate possible workarounds.« less

Increased pentosidine, an advanced glycation end product, in serum and synovial fluid from patients with knee osteoarthritis and its relation with cartilage oligomeric matrix protein

PubMed Central

Senolt, L; Braun, M; Olejarova, M; Forejtova, S; Gatterova, J; Pavelka, K

2005-01-01

Background: Pentosidine, an advanced glycation end product, increasingly accumulates in articular cartilage with age, and contributes to the pathogenesis of osteoarthritis (OA). Increased pentosidine concentrations are associated with inflammatory disorders—for example, rheumatoid arthritis. Objective: To compare pentosidine serum concentrations in patients with knee OA and in healthy volunteers and to determine a relationship between pentosidine and cartilage oligomeric matrix protein (COMP)—a marker of articular cartilage destruction. Methods: Paired serum and synovial fluid samples were obtained by arthrocentesis from 38 patients with knee OA and from 38 healthy volunteers. Pentosidine concentration was measured by reverse phase high performance liquid chromatography with fluorescent detection and COMP was determined by sandwich ELISA. Results: Significantly increased serum pentosidine (p<0.01) and COMP (p<0.05) levels were detected in the patients with OA compared with the control group. Serum pentosidine correlated significantly with synovial fluid pentosidine (p<0.001). Pentosidine in synovial fluid (p<0.05) and in serum (p<0.05) correlated significantly with synovial fluid COMP. Pentosidine and COMP concentrations did not correlate significantly with the radiological stage of the disease. Conclusion: Increased pentosidine serum concentration in patients with OA and its correlation with the cartilage destruction marker COMP in synovial fluid suggests that pentosidine may be important in OA pathology and is a new potential OA marker. PMID:15897309

Resolving Fast, Confined Diffusion in Bacteria with Image Correlation Spectroscopy.

PubMed

Rowland, David J; Tuson, Hannah H; Biteen, Julie S

2016-05-24

By following single fluorescent molecules in a microscope, single-particle tracking (SPT) can measure diffusion and binding on the nanometer and millisecond scales. Still, although SPT can at its limits characterize the fastest biomolecules as they interact with subcellular environments, this measurement may require advanced illumination techniques such as stroboscopic illumination. Here, we address the challenge of measuring fast subcellular motion by instead analyzing single-molecule data with spatiotemporal image correlation spectroscopy (STICS) with a focus on measurements of confined motion. Our SPT and STICS analysis of simulations of the fast diffusion of confined molecules shows that image blur affects both STICS and SPT, and we find biased diffusion rate measurements for STICS analysis in the limits of fast diffusion and tight confinement due to fitting STICS correlation functions to a Gaussian approximation. However, we determine that with STICS, it is possible to correctly interpret the motion that blurs single-molecule images without advanced illumination techniques or fast cameras. In particular, we present a method to overcome the bias due to image blur by properly estimating the width of the correlation function by directly calculating the correlation function variance instead of using the typical Gaussian fitting procedure. Our simulation results are validated by applying the STICS method to experimental measurements of fast, confined motion: we measure the diffusion of cytosolic mMaple3 in living Escherichia coli cells at 25 frames/s under continuous illumination to illustrate the utility of STICS in an experimental parameter regime for which in-frame motion prevents SPT and tight confinement of fast diffusion precludes stroboscopic illumination. Overall, our application of STICS to freely diffusing cytosolic protein in small cells extends the utility of single-molecule experiments to the regime of fast confined diffusion without requiring advanced microscopy techniques. Copyright © 2016 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Towards sensitive, high-throughput, biomolecular assays based on fluorescence lifetime

NASA Astrophysics Data System (ADS)

Ioanna Skilitsi, Anastasia; Turko, Timothé; Cianfarani, Damien; Barre, Sophie; Uhring, Wilfried; Hassiepen, Ulrich; Léonard, Jérémie

2017-09-01

Time-resolved fluorescence detection for robust sensing of biomolecular interactions is developed by implementing time-correlated single photon counting in high-throughput conditions. Droplet microfluidics is used as a promising platform for the very fast handling of low-volume samples. We illustrate the potential of this very sensitive and cost-effective technology in the context of an enzymatic activity assay based on fluorescently-labeled biomolecules. Fluorescence lifetime detection by time-correlated single photon counting is shown to enable reliable discrimination between positive and negative control samples at a throughput as high as several hundred samples per second.

Differences in fluorescence profiles from breast cancer tissues due to changes in relative tryptophan content via energy transfer: tryptophan content correlates with histologic grade and tumor size but not with lymph node metastases

NASA Astrophysics Data System (ADS)

Sordillo, Laura A.; Sordillo, Peter P.; Budansky, Yury; Pu, Yang; Alfano, Robert R.

2014-12-01

The correlation between histologic grade, an increasingly important measure of prognosis for patients with breast cancer, and tryptophan levels from tissues of 15 breast carcinoma patients was investigated. Changes in the relative content of key native organic biomolecule tryptophan were seen from the fluorescence spectra of cancerous and paired normal tissues with excitation wavelengths of 280 and 300 nm. Due to a large spectral overlap and matching excitation-emission spectra, fluorescence resonance energy transfer from tryptophan-donor to reduced nicotinamide adenine dinucleotides-acceptor was noted. We used the ratios of fluorescence intensities at their spectral emission peaks, or spectral fingerprint peaks, at 340, 440, and 460 nm. Higher ratios correlated strongly with high histologic grade, while lower-grade tumors had low ratios. Large tumor size also correlated with high ratios, while the number of lymph node metastases, a major factor in staging, was not correlated with tryptophan levels. High histologic grade correlates strongly with increased content of tryptophan in breast cancer tissues and suggests that measurement of tryptophan content may be useful as a part of the evaluation of these patients.

Advances in Surface-Enhanced Fluorescence

PubMed Central

Lakowicz, Joseph R.; Geddes, Chris D.; Gryczynski, Ignacy; Malicka, Joanna; Gryczynski, Zygmunt; Aslan, Kadir; Lukomska, Joanna; Matveeva, Evgenia; Zhang, Jian; Badugu, Ramachandram; Huang, Jun

2009-01-01

We report recent achievements in metal-enhanced fluorescence from our laboratory. Several fluorophore systems have been studied on metal particle-coated surfaces and in colloid suspensions. In particular, we describe a distance dependent enhancement on silver island films (SIFs), release of self-quenching of fluorescence near silver particles, and the applications of fluorescence enhancement near metalized surfaces to bioassays. We discuss a number of methods for various shaped silver particle deposition on surfaces. PMID:15617385

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.

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).

Plant stress detection by remote measurement of fluorescence

USGS Publications Warehouse

McFarlane, J. C.; Watson, Robert D.; Theisen, Arnold F.; Jackson, R. D.; Ehrler, W. L.; Pinter, P. J.; Idso, S. B.; Reginato, R. J.

1980-01-01

Chlorophyll fluorescence of mature lemon trees was measured with a Fraunhofer line discriminator (FLD). An increase in fluorescence was correlated with plant water stress as measured by stomatal resistance and twig water potential.

Position-sensitive scanning fluorescence correlation spectroscopy.

PubMed

Skinner, Joseph P; Chen, Yan; Müller, Joachim D

2005-08-01

Fluorescence correlation spectroscopy (FCS) uses a stationary laser beam to illuminate a small sample volume and analyze the temporal behavior of the fluorescence fluctuations within the stationary observation volume. In contrast, scanning FCS (SFCS) collects the fluorescence signal from a moving observation volume by scanning the laser beam. The fluctuations now contain both temporal and spatial information about the sample. To access the spatial information we synchronize scanning and data acquisition. Synchronization allows us to evaluate correlations for every position along the scanned trajectory. We use a circular scan trajectory in this study. Because the scan radius is constant, the phase angle is sufficient to characterize the position of the beam. We introduce position-sensitive SFCS (PSFCS), where correlations are calculated as a function of lag time and phase. We present the theory of PSFCS and derive expressions for diffusion, diffusion in the presence of flow, and for immobilization. To test PSFCS we compare experimental data with theory. We determine the direction and speed of a flowing dye solution and the position of an immobilized particle. To demonstrate the feasibility of the technique for applications in living cells we present data of enhanced green fluorescent protein measured in the nucleus of COS cells.

Colocalization analysis in fluorescence micrographs: verification of a more accurate calculation of pearson's correlation coefficient.

PubMed

Barlow, Andrew L; Macleod, Alasdair; Noppen, Samuel; Sanderson, Jeremy; Guérin, Christopher J

2010-12-01

One of the most routine uses of fluorescence microscopy is colocalization, i.e., the demonstration of a relationship between pairs of biological molecules. Frequently this is presented simplistically by the use of overlays of red and green images, with areas of yellow indicating colocalization of the molecules. Colocalization data are rarely quantified and can be misleading. Our results from both synthetic and biological datasets demonstrate that the generation of Pearson's correlation coefficient between pairs of images can overestimate positive correlation and fail to demonstrate negative correlation. We have demonstrated that the calculation of a thresholded Pearson's correlation coefficient using only intensity values over a determined threshold in both channels produces numerical values that more accurately describe both synthetic datasets and biological examples. Its use will bring clarity and accuracy to colocalization studies using fluorescent microscopy.

The Potential Applications of Real-Time Monitoring of Water Quality in a Large Shallow Lake (Lake Taihu, China) Using a Chromophoric Dissolved Organic Matter Fluorescence Sensor

PubMed Central

Niu, Cheng; Zhang, Yunlin; Zhou, Yongqiang; Shi, Kun; Liu, Xiaohan; Qin, Boqiang

2014-01-01

This study presents results from field surveys performed over various seasons in a large, eutrophic, shallow lake (Lake Taihu, China) using an in situ chromophoric dissolved organic matter (CDOM) fluorescence sensor as a surrogate for other water quality parameters. These measurements identified highly significant empirical relationships between CDOM concentration measured using the in situ fluorescence sensor and CDOM absorption, fluorescence, dissolved organic carbon (DOC), chemical oxygen demand (COD) and total phosphorus (TP) concentrations. CDOM concentration expressed in quinine sulfate equivalent units, was highly correlated with the CDOM absorption coefficient (r2 = 0.80, p < 0.001), fluorescence intensities (Ex./Em. 370/460 nm) (r2 = 0.91, p < 0.001), the fluorescence index (r2 = 0.88, p < 0.001) and the humification index (r2 = 0.78, p < 0.001), suggesting that CDOM concentration measured using the in situ fluorescence sensor could act as a substitute for the CDOM absorption coefficient and fluorescence measured in the laboratory. Similarly, CDOM concentration was highly correlated with DOC concentration (r2 = 0.68, p < 0.001), indicating that in situ CDOM fluorescence sensor measurements could be a proxy for DOC concentration. In addition, significant positive correlations were found between laboratory CDOM absorption coefficients and COD (r2 = 0.83, p < 0.001), TP (r2 = 0.82, p < 0.001) concentrations, suggesting a potential further application for the real-time monitoring of water quality using an in situ CDOM fluorescence sensor. PMID:24984060

The potential applications of real-time monitoring of water quality in a large shallow lake (Lake Taihu, China) using a chromophoric dissolved organic matter fluorescence sensor.

PubMed

Niu, Cheng; Zhang, Yunlin; Zhou, Yongqiang; Shi, Kun; Liu, Xiaohan; Qin, Boqiang

2014-06-30

This study presents results from field surveys performed over various seasons in a large, eutrophic, shallow lake (Lake Taihu, China) using an in situ chromophoric dissolved organic matter (CDOM) fluorescence sensor as a surrogate for other water quality parameters. These measurements identified highly significant empirical relationships between CDOM concentration measured using the in situ fluorescence sensor and CDOM absorption, fluorescence, dissolved organic carbon (DOC), chemical oxygen demand (COD) and total phosphorus (TP) concentrations. CDOM concentration expressed in quinine sulfate equivalent units, was highly correlated with the CDOM absorption coefficient (r(2) = 0.80, p < 0.001), fluorescence intensities (Ex./Em. 370/460 nm) (r(2) = 0.91, p < 0.001), the fluorescence index (r(2) = 0.88, p < 0.001) and the humification index (r(2) = 0.78, p < 0.001), suggesting that CDOM concentration measured using the in situ fluorescence sensor could act as a substitute for the CDOM absorption coefficient and fluorescence measured in the laboratory. Similarly, CDOM concentration was highly correlated with DOC concentration (r(2) = 0.68, p < 0.001), indicating that in situ CDOM fluorescence sensor measurements could be a proxy for DOC concentration. In addition, significant positive correlations were found between laboratory CDOM absorption coefficients and COD (r(2) = 0.83, p < 0.001), TP (r(2) = 0.82, p < 0.001) concentrations, suggesting a potential further application for the real-time monitoring of water quality using an in situ CDOM fluorescence sensor.

Quantitative confocal fluorescence microscopy of dynamic processes by multifocal fluorescence correlation spectroscopy

NASA Astrophysics Data System (ADS)

Krmpot, Aleksandar J.; Nikolić, Stanko N.; Vitali, Marco; Papadopoulos, Dimitrios K.; Oasa, Sho; Thyberg, Per; Tisa, Simone; Kinjo, Masataka; Nilsson, Lennart; Gehring, Walter J.; Terenius, Lars; Rigler, Rudolf; Vukojevic, Vladana

2015-07-01

Quantitative confocal fluorescence microscopy imaging without scanning is developed for the study of fast dynamical processes. The method relies on the use of massively parallel Fluorescence Correlation Spectroscopy (mpFCS). Simultaneous excitation of fluorescent molecules across the specimen is achieved by passing a single laser beam through a Diffractive Optical Element (DOE) to generate a quadratic illumination matrix of 32×32 light sources. Fluorescence from 1024 illuminated spots is detected in a confocal arrangement by a matching matrix detector consisting of the same number of single-photon avalanche photodiodes (SPADs). Software was developed for data acquisition and fast autoand cross-correlation analysis by parallel signal processing using a Graphic Processing Unit (GPU). Instrumental performance was assessed using a conventional single-beam FCS instrument as a reference. Versatility of the approach for application in biomedical research was evaluated using ex vivo salivary glands from Drosophila third instar larvae expressing a fluorescently-tagged transcription factor Sex Combs Reduced (Scr) and live PC12 cells stably expressing the fluorescently tagged mu-opioid receptor (MOPeGFP). We show that quantitative mapping of local concentration and mobility of transcription factor molecules across the specimen can be achieved using this approach, which paves the way for future quantitative characterization of dynamical reaction-diffusion landscapes across live cells/tissue with a submillisecond temporal resolution (presently 21 μs/frame) and single-molecule sensitivity.

Developing LED UV fluorescence sensors for online monitoring DOM and predicting DBPs formation potential during water treatment.

PubMed

Li, Wen-Tao; Jin, Jing; Li, Qiang; Wu, Chen-Fei; Lu, Hai; Zhou, Qing; Li, Ai-Min

2016-04-15

Online monitoring dissolved organic matter (DOM) is urgent for water treatment management. In this study, high performance size exclusion chromatography with multi-UV absorbance and multi-emission fluorescence scans were applied to spectrally characterize samples from 16 drinking water sources across Yangzi River and Huai River Watersheds. The UV absorbance indices at 254 nm and 280 nm referred to the same DOM components and concentration, and the 280 nm UV light could excite both protein-like and humic-like fluorescence. Hence a novel UV fluorescence sensor was developed out using only one UV280 light-emitting diode (LED) as light source. For all samples, enhanced coagulation was mainly effective for large molecular weight biopolymers; while anion exchange further substantially removed humic substances. During chlorination tests, UVA280 and UVA254 showed similar correlations with yields of disinfection byproducts (DBPs); the humic-like fluorescence obtained from LED sensors correlated well with both trihalomethanes and haloacetic acids yields, while the correlation between protein-like fluorescence and trihalomethanes was relatively poor. Anion exchange exhibited more reduction of DBPs yields as well as UV absorbance and fluorescence signals than enhanced coagulation. The results suggest that the LED UV fluorescence sensors are very promising for online monitoring DOM and predicting DBPs formation potential during water treatment. Copyright © 2016 Elsevier Ltd. All rights reserved.

Correlative cryogenic tomography of cells using light and soft x-rays

PubMed Central

Smith, Elizabeth A.; Cinquin, Bertrand P.; Do, Myan; McDermott, Gerry; Le Gros, Mark A.; Larabell, Carolyn A.

2013-01-01

Correlated imaging is the process of imaging a specimen with two complementary modalities, and then combining the two data sets to create a highly informative, composite view. A recent implementation of this concept has been the combination of soft x-ray tomography (SXT) with fluorescence cryogenic microscopy (FCM). SXT-FCM is used to visualize cells that are held in a near-native, cryo-preserved state. The resultant images are, therefore, highly representative of both the cellular architecture and molecular organization in vivo. SXT quantitatively visualizes the cell and sub-cellular structures; FCM images the spatial distribution of fluorescently labeled molecules. Here, we review the characteristics of SXT-FCM, and briefly discuss how this method compares with existing correlative imaging techniques. We also describe how the incorporation of a cryo-rotation stage into a cryogenic fluorescence microscope allows acquisition of fluorescence cryogenic tomography (FCT) data. FCT is optimally suited to correlation with SXT, since both techniques image the specimen in 3-D, potentially with similar, isotropic spatial resolution. PMID:24355261

Recent Advances in Fluorescent Angioscopy for Molecular Imaging of Human Atherosclerotic Coronary Plaque

PubMed Central

2017-01-01

Purpose of Review: In vivo imaging of the native substances, including lipoproteins, that comprise human atherosclerotic plaques is currently beyond the scope of any available imaging techniques. Color and near-infrared fluorescent angioscopy (CFA and NIRFA, respectively) systems have been recently developed for molecular imaging of lipoproteins within the human coronary arterial wall ex vivo and/or in vivo. The author reviews recent findings on lipoprotein deposition in human coronary plaques obtained by these imaging techniques. Recent Findings: Using specific biomarkers, native pro-atherogenic substances such as oxidized low-density lipoprotein (ox-LDL), LDL, triglycerides (TG), apolipoprotein B-100 (ApoB-100), and lysophosphatidylcholine (LPC), and the anti-atherogenic substance such as high-density lipoprotein (HDL) were visualized by CFA, and LDL and cholesterol by NIRFA, in coronary plaques obtained from autopsy subjects. The relationship between incidence and plaque morphology differed for each substance. The incidence of ox-LDL and LDL on color fluorescence microscopy correlated well with that observed using immunohistochemical techniques. During coronary catheterization in patients, ox-LDL, LDL, and HDL in coronary plaques were visualized by CFA or NIRFA. Conclusions: Using CFA or NIRFA, the distribution of the major native pro-atherogenic and antiatherogenic lipoproteins and their components within human coronary plaques can be evaluated ex vivo and/or in vivo. Fluorescent angioscopy could help our understanding of the molecular mechanisms of coronary atherosclerosis and in the evaluation of the effects of therapy targeting the substances comprising atherosclerotic coronary plaques. PMID:28381766

Near Infrared Dyes as Lifetime Solvatochromic Probes for Micropolarity Measurements of Biological Systems

PubMed Central

Berezin, Mikhail Y.; Lee, Hyeran; Akers, Walter; Achilefu, Samuel

2007-01-01

The polarity of biological mediums controls a host of physiological processes such as digestion, signaling, transportation, metabolism, and excretion. With the recent widespread use of near-infrared (NIR) fluorescent dyes for biological imaging of cells and living organisms, reporting medium polarity with these dyes would provide invaluable functional information in addition to conventional optical imaging parameters. Here, we report a new approach to determine polarities of macro- and microsystems for in vitro and potential in vivo applications using NIR polymethine molecular probes. Unlike the poor solvatochromic response of NIR dyes in solvents with diverse polarity, their fluorescence lifetimes are highly sensitive, increasing by a factor of up to 8 on moving from polar to nonpolar mediums. We also established a correlation between fluorescence lifetime and solvent orientation polarizability and developed a lifetime polarity index for determining the polarity of complex systems, including micelles and albumin binding sites. Because of the importance of medium polarity in molecular, cellular, and biochemical processes and the significance of reduced autofluorescence and deep tissue penetration of light in the NIR region, the findings reported herein represent an important advance toward using NIR molecular probes to measure the polarity of complex biological systems in vitro and in vivo. PMID:17573433

A portable microscopy system for fluorescence, polarized, and brightfield imaging

NASA Astrophysics Data System (ADS)

Gordon, Paul; Wattinger, Rolla; Lewis, Cody; Venancio, Vinicius Paula; Mertens-Talcott, Susanne U.; Coté, Gerard

2018-02-01

The use of mobile phones to conduct diagnostic microscopy at the point-of-care presents intriguing possibilities for the advancement of high-quality medical care in remote settings. However, it is challenging to create a single device that can adapt to the ever-varying camera technologies in phones or that can image with the customization that multiple modalities require for applications such as malaria diagnosis. A portable multi-modal microscope system is presented that utilizes a Raspberry Pi to collect and transmit data wirelessly to a myriad of electronic devices for image analysis. The microscopy system is capable of providing to the user correlated brightfield, polarized, and fluorescent images of samples fixed on traditional microscopy slides. The multimodal diagnostic capabilities of the microscope were assessed by measuring parasitemia of Plasmodium falciparum-infected thin blood smears. The device is capable of detecting fluorescently-labeled DNA using FITC excitation (490 nm) and emission (525 nm), the birefringent P. falciparum byproduct hemozoin, and detecting brightfield absorption with a resolution of 0.78 micrometers (element 9-3 of a 1951 Air Force Target). This microscopy system is a novel portable imaging tool that may be a viable candidate for field implementation if challenges of system durability, cost considerations, and full automation can be overcome.

New hardware and workflows for semi-automated correlative cryo-fluorescence and cryo-electron microscopy/tomography.

PubMed

Schorb, Martin; Gaechter, Leander; Avinoam, Ori; Sieckmann, Frank; Clarke, Mairi; Bebeacua, Cecilia; Bykov, Yury S; Sonnen, Andreas F-P; Lihl, Reinhard; Briggs, John A G

2017-02-01

Correlative light and electron microscopy allows features of interest defined by fluorescence signals to be located in an electron micrograph of the same sample. Rare dynamic events or specific objects can be identified, targeted and imaged by electron microscopy or tomography. To combine it with structural studies using cryo-electron microscopy or tomography, fluorescence microscopy must be performed while maintaining the specimen vitrified at liquid-nitrogen temperatures and in a dry environment during imaging and transfer. Here we present instrumentation, software and an experimental workflow that improves the ease of use, throughput and performance of correlated cryo-fluorescence and cryo-electron microscopy. The new cryo-stage incorporates a specially modified high-numerical aperture objective lens and provides a stable and clean imaging environment. It is combined with a transfer shuttle for contamination-free loading of the specimen. Optimized microscope control software allows automated acquisition of the entire specimen area by cryo-fluorescence microscopy. The software also facilitates direct transfer of the fluorescence image and associated coordinates to the cryo-electron microscope for subsequent fluorescence-guided automated imaging. Here we describe these technological developments and present a detailed workflow, which we applied for automated cryo-electron microscopy and tomography of various specimens. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

Automated sub-5 nm image registration in integrated correlative fluorescence and electron microscopy using cathodoluminescence pointers

NASA Astrophysics Data System (ADS)

Haring, Martijn T.; Liv, Nalan; Zonnevylle, A. Christiaan; Narvaez, Angela C.; Voortman, Lenard M.; Kruit, Pieter; Hoogenboom, Jacob P.

2017-03-01

In the biological sciences, data from fluorescence and electron microscopy is correlated to allow fluorescence biomolecule identification within the cellular ultrastructure and/or ultrastructural analysis following live-cell imaging. High-accuracy (sub-100 nm) image overlay requires the addition of fiducial markers, which makes overlay accuracy dependent on the number of fiducials present in the region of interest. Here, we report an automated method for light-electron image overlay at high accuracy, i.e. below 5 nm. Our method relies on direct visualization of the electron beam position in the fluorescence detection channel using cathodoluminescence pointers. We show that image overlay using cathodoluminescence pointers corrects for image distortions, is independent of user interpretation, and does not require fiducials, allowing image correlation with molecular precision anywhere on a sample.

Automated sub-5 nm image registration in integrated correlative fluorescence and electron microscopy using cathodoluminescence pointers.

PubMed

Haring, Martijn T; Liv, Nalan; Zonnevylle, A Christiaan; Narvaez, Angela C; Voortman, Lenard M; Kruit, Pieter; Hoogenboom, Jacob P

2017-03-02

In the biological sciences, data from fluorescence and electron microscopy is correlated to allow fluorescence biomolecule identification within the cellular ultrastructure and/or ultrastructural analysis following live-cell imaging. High-accuracy (sub-100 nm) image overlay requires the addition of fiducial markers, which makes overlay accuracy dependent on the number of fiducials present in the region of interest. Here, we report an automated method for light-electron image overlay at high accuracy, i.e. below 5 nm. Our method relies on direct visualization of the electron beam position in the fluorescence detection channel using cathodoluminescence pointers. We show that image overlay using cathodoluminescence pointers corrects for image distortions, is independent of user interpretation, and does not require fiducials, allowing image correlation with molecular precision anywhere on a sample.

Automated sub-5 nm image registration in integrated correlative fluorescence and electron microscopy using cathodoluminescence pointers

PubMed Central

Haring, Martijn T.; Liv, Nalan; Zonnevylle, A. Christiaan; Narvaez, Angela C.; Voortman, Lenard M.; Kruit, Pieter; Hoogenboom, Jacob P.

2017-01-01

In the biological sciences, data from fluorescence and electron microscopy is correlated to allow fluorescence biomolecule identification within the cellular ultrastructure and/or ultrastructural analysis following live-cell imaging. High-accuracy (sub-100 nm) image overlay requires the addition of fiducial markers, which makes overlay accuracy dependent on the number of fiducials present in the region of interest. Here, we report an automated method for light-electron image overlay at high accuracy, i.e. below 5 nm. Our method relies on direct visualization of the electron beam position in the fluorescence detection channel using cathodoluminescence pointers. We show that image overlay using cathodoluminescence pointers corrects for image distortions, is independent of user interpretation, and does not require fiducials, allowing image correlation with molecular precision anywhere on a sample. PMID:28252673

Detection of rheumatoid arthritis by evaluation of normalized variances of fluorescence time correlation functions

NASA Astrophysics Data System (ADS)

Dziekan, Thomas; Weissbach, Carmen; Voigt, Jan; Ebert, Bernd; MacDonald, Rainer; Bahner, Malte L.; Mahler, Marianne; Schirner, Michael; Berliner, Michael; Berliner, Birgitt; Osel, Jens; Osel, Ilka

2011-07-01

Fluorescence imaging using the dye indocyanine green as a contrast agent was investigated in a prospective clinical study for the detection of rheumatoid arthritis. Normalized variances of correlated time series of fluorescence intensities describing the bolus kinetics of the contrast agent in certain regions of interest were analyzed to differentiate healthy from inflamed finger joints. These values are determined using a robust, parameter-free algorithm. We found that the normalized variance of correlation functions improves the differentiation between healthy joints of volunteers and joints with rheumatoid arthritis of patients by about 10% compared to, e.g., ratios of areas under the curves of raw data.

Measurement of protein-like fluorescence in river and waste water using a handheld spectrophotometer.

PubMed

Baker, Andy; Ward, David; Lieten, Shakti H; Periera, Ryan; Simpson, Ellie C; Slater, Malcolm

2004-07-01

Protein-like fluorescence intensity in rivers increases with increasing anthropogenic DOM inputs from sewerage and farm wastes. Here, a portable luminescence spectrophotometer was used to investigate if this technology could be used to provide both field scientists with a rapid pollution monitoring tool and process control engineers with a portable waste water monitoring device, through the measurement of river and waste water tryptophan-like fluorescence from a range of rivers in NE England and from effluents from within two waste water treatment plants. The portable spectrophotometer determined that waste waters and sewerage effluents had the highest tryptophan-like fluorescence intensity, urban streams had an intermediate tryptophan-like fluorescence intensity, and the upstream river samples of good water quality the lowest tryptophan-like fluorescence intensity. Replicate samples demonstrated that fluorescence intensity is reproducible to +/- 20% for low fluorescence, 'clean' river water samples and +/- 5% for urban water and waste waters. Correlations between fluorescence measured by the portable spectrophotometer with a conventional bench machine were 0.91; (Spearman's rho, n = 143), demonstrating that the portable spectrophotometer does correlate with tryptophan-like fluorescence intensity measured using the bench spectrophotometer.

Probing Cytoskeletal Structures by Coupling Optical Superresolution and AFM Techniques for a Correlative Approach

PubMed Central

Chacko, Jenu Varghese; Zanacchi, Francesca Cella; Diaspro, Alberto

2013-01-01

In this article, we describe and show the application of some of the most advanced fluorescence superresolution techniques, STED AFM and STORM AFM microscopy towards imaging of cytoskeletal structures, such as microtubule filaments. Mechanical and structural properties can play a relevant role in the investigation of cytoskeletal structures of interest, such as microtubules, that provide support to the cell structure. In fact, the mechanical properties, such as the local stiffness and the elasticity, can be investigated by AFM force spectroscopy with tens of nanometers resolution. Force curves can be analyzed in order to obtain the local elasticity (and the Young's modulus calculation by fitting the force curves from every pixel of interest), and the combination with STED/STORM microscopy integrates the measurement with high specificity and yields superresolution structural information. This hybrid modality of superresolution-AFM working is a clear example of correlative multimodal microscopy. PMID:24027190

Quantitative evaluation of cross correlation between two finite-length time series with applications to single-molecule FRET.

PubMed

Hanson, Jeffery A; Yang, Haw

2008-11-06

The statistical properties of the cross correlation between two time series has been studied. An analytical expression for the cross correlation function's variance has been derived. On the basis of these results, a statistically robust method has been proposed to detect the existence and determine the direction of cross correlation between two time series. The proposed method has been characterized by computer simulations. Applications to single-molecule fluorescence spectroscopy are discussed. The results may also find immediate applications in fluorescence correlation spectroscopy (FCS) and its variants.

Correlative fluorescence and electron microscopy of quantum dot labeled proteins on whole cells in liquid.

PubMed

Peckys, Diana B; Dukes, Madeline J; de Jonge, Niels

2014-01-01

Correlative fluorescence microscopy and scanning transmission electron microscopy (STEM) of cells fully immersed in liquid is a new methodology with many application areas. Proteins, in live cells immobilized on microchips, are labeled with fluorescent quantum dot (QD) nanoparticles. In this protocol, the epidermal growth factor receptor (EGFR) is labeled. The cells are fixed after a selected labeling time, for example, 5 min as needed to form EGFR dimers. The microchip with cells is then imaged with fluorescence microscopy. Thereafter, the microchip with the labeled cells and one with a spacer are assembled in a special microfluidic device and imaged with STEM.

Fluorescence from the maillard reaction and its potential applications in food science.

PubMed

Matiacevich, Silvia B; Santagapita, Patricio R; Buera, M Pilar

2005-01-01

The chemistry of the Maillard reaction involves a complex set of steps, and its interpretation represents a challenge in basic and applied aspects of Food Science. Fluorescent compounds have been recognized as important early markers of the reaction in food products since 1942. However, the recent advances in the characterization of fluorophores' development were observed in biological and biomedical areas. The in vivo non-enzymatic glycosylation of proteins produces biological effects, promoting health deterioration. The characteristic fluorescence of advanced glycosylation end products (AGEs) is similar to that of Maillard food products and represents an indicator of the level of AGE-modified proteins, but the structure of the fluorescent groups is, typically, unknown. Application of fluorescence measurement is considered a potential tool for addressing key problems of food deterioration as an early marker or index of the damage of biomolecules. Fluorophores may be precursors of the brown pigments and/or end products. A general scheme of the Maillard reaction is proposed in this article, incorporating the pool concept. A correct interpretation of the effect of environmental and compositional conditions and their influences on the reaction kinetics may help to define the meaning of fluorescence development for each particular system.

Correlative Fluorescence and Electron Microscopy in 3D-Scanning Electron Microscope Perspective.

PubMed

Franks, Jonathan; Wallace, Callen T; Shibata, Masateru; Suga, Mitsuo; Erdman, Natasha; Stolz, Donna B; Watkins, Simon C

2017-04-03

The ability to correlate fluorescence microscopy (FM) and electron microscopy (EM) data obtained on biological (cell and tissue) specimens is essential to bridge the resolution gap between the data obtained by these different imaging techniques. In the past such correlations were limited to either EM navigation in two dimensions to the locations previously highlighted by fluorescence markers, or subsequent high-resolution acquisition of tomographic information using a TEM. We present a novel approach whereby a sample previously investigated by FM is embedded and subjected to sequential mechanical polishing and backscatter imaging by scanning electron microscope. The resulting three dimensional EM tomogram of the sample can be directly correlated to the FM data. © 2017 by John Wiley & Sons, Inc. Copyright © 2017 John Wiley & Sons, Inc.

Fluorescence correlation spectroscopy: the case of subdiffusion.

PubMed

Lubelski, Ariel; Klafter, Joseph

2009-03-18

The theory of fluorescence correlation spectroscopy is revisited here for the case of subdiffusing molecules. Subdiffusion is assumed to stem from a continuous-time random walk process with a fat-tailed distribution of waiting times and can therefore be formulated in terms of a fractional diffusion equation (FDE). The FDE plays the central role in developing the fluorescence correlation spectroscopy expressions, analogous to the role played by the simple diffusion equation for regular systems. Due to the nonstationary nature of the continuous-time random walk/FDE, some interesting properties emerge that are amenable to experimental verification and may help in discriminating among subdiffusion mechanisms. In particular, the current approach predicts 1), a strong dependence of correlation functions on the initial time (aging); 2), sensitivity of correlation functions to the averaging procedure, ensemble versus time averaging (ergodicity breaking); and 3), that the basic mean-squared displacement observable depends on how the mean is taken.

"Sizing" the oligomers of Azami Green fluorescent protein with FCS and antibunching

NASA Astrophysics Data System (ADS)

Temirov, Jamshid; Werner, James H.; Goodwin, Peter M.; Bradbury, Andrew R. M.

2012-02-01

Fluorescent proteins are invaluable molecules in fluorescence microscopy and spectroscopy. The size and brightness of fluorescent proteins often dictates the application they may be used for. While a monomeric protein may be the least perturbative structure for labeling a protein in a cell, often oligomers (dimers and tetramers) of fluorescent proteins can be more stable. However, from a quantitative microscopy standpoint, it is important to realize the photophysical properties of monomers do not necessarily multiply by their number when they form oligomers. In this work we studied oligomerization states of the Azami Green (AG) protein with fluorescence correlation spectroscopy (FCS) and photon antibunching or photon pair correlation spectroscopy (PPCS). FCS was used to measure the hydrodynamic size of the oligomers, whereas antibunching was used to count the number of fluorescent emitters in the oligomers. The results exhibited that the dimers of AG were single emitters and the tetramers were dual-emitters, indicative of dipole-dipole interactions and energy transfer between the monomeric units. We also used these methods to estimate the number of fluorescent proteins displayed on T7 phage molecules.

Differential fluorescence EEMs can be used to assess treatability of DOM during drinking water production

NASA Astrophysics Data System (ADS)

Lavonen, Elin; Kothawala, Dolly; Tranvik, Lars; Köhler, Stephan

2014-05-01

Fluorescence spectroscopy has been widely used to characterize fluorescent dissolved organic matter (FDOM) in various waters including during drinking water production. Commonly used techniques for data treatment include peak picking, indexes calculated from 2D emission spectra and modelling of fluorescence components using parallel factor analysis (PARAFAC). However, peak picking and indexes only use limited information from the fluorescence EEMs and PARAFAC requires a larger dataset and experience to perform. Because DOM is a major issue in drinking water production, and personnel at water treatment plants usually have limited time for advanced analysis we have developed a simple way of assessing the treatability of DOM in different waters using differential fluorescence. With this approach the removed fraction of FDOM is calculated from samples taken before and after a particular treatment process and the percentage of removed material assessed. Samples have been collected from four large water treatment plants in Sweden and analyzed for 3Dfluorescence, absorbance and DOC. The selective removal of DOM during e.g. flocculation and slow sand filtration as well as differences in experienced treatability between the treatment plants was described with differential fluorescence. Chemical flocculation is selective towards FDOM with red-shifted emission across the entire EEM. Red-shift has earlier been connected to condensation (i.e. decrease in H/C) and positively correlated to molecular size indicating that larger, humified molecules are being preferentially removed. During the biological process of slow sand filtration compounds with blue-shifted emission are targeted demonstrating selective removal of more freshly produced, microbial material. Disinfection with UV/NH2Cl and NaOCl was found to only target material with protein-like fluorescence suggesting that FDOM of this nature could be responsible for unwanted consumption of disinfection agent. Targeted removal of this fraction prior to disinfection should optimize the process. Furthermore, the main process at all studied WTPs is flocculation and their experienced treatability could easily be explained through the percentage of FDOM with emission above 450 nm (p<0.0001).

Steady-state chlorophyll fluorescence (Fs) measurements as a tool to follow variations of net CO2 assimilation and stomatal conductance during water-stress in C3 plants.

PubMed

Flexas, Jaume; Escalona, José Mariano; Evain, Sebastian; Gulías, Javier; Moya, Ismaël; Osmond, Charles Barry; Medrano, Hipólito

2002-02-01

Water stress experiments were performed with grapevines (Vitis vinifera L.) and other C3 plants in the field, in potted plants in the laboratory, and with detached leaves. It was found that, in all cases, the ratio of steady state chlorophyll fluorescence (Fs) normalized to dark-adapted intrinsic fluorescence (Fo) inversely correlated with non-photochemical quenching (NPQ). Also, at high irradiance, the ratio Fs/Fo was positively correlated with CO2 assimilation in air, with electron transport rate calculated from fluorescence, and with stomatal conductance, but no clear correlation was observed with qP. The significance of these relationships is discussed. The ratio Fs/Fo, measured with a portable instrument (PAM-2000) or with a remote sensing FIPAM system, provides a good method for the early detection of water stress, and may become a useful guide to irrigation requirements.

Differences in Non-Enzymatic Glycation and Collagen Crosslinks between Human Cortical and Cancellous Bone

PubMed Central

Karim, Lamya; Tang, Simon Y.; Sroga, Grażyna E.; Vashishth, Deepak

2015-01-01

Purpose Accumulation of collagen crosslinks (advanced glycation end products [AGEs]) produced by non-enzymatic glycation deteriorates bone's mechanical properties and fracture resistance. Although a single AGE, pentosidine, is commonly used as a representative marker, it is unclear whether it quantitatively reflects total fluorescent AGEs in bone. The goal of this study was to establish the relationship between pentosidine and total AGEs in cancellous and cortical bone. Methods Pentosidine and total AGEs were quantified in 170 human bone samples. Total fluorescent AGEs were measured in 28 additional cancellous and cortical bone specimens of the same apparent volume that were incubated in control or in vitro glycation solutions. Correlations between pentosidine and total AGEs and differences between cortical and cancellous groups were determined. Results Pentosidine was correlated with total AGEs in cancellous bone (r=0.53, p<0.0001) and weakly correlated in cortical bone (r=0.23, p<0.05). There was more pentosidine (p<0.01) and total AGEs (p<0.001) in cancellous than in cortical bone. The in vitro glycation sub-study showed that cancellous bone accumulated more AGEs than cortical bone (p<0.05). Conclusion The relationship between pentosidine and total AGEs and their magnitude of accumulation differed in cancellous and cortical bone of the same apparent volume, and were dependent on the surface-to-volume ratios of each sample. It is important to consider the bone types as two separate entities, and it is crucial to quantify total AGEs in addition to pentosidine to allow for more comprehensive analysis of the effects of non-enzymatic glycation in bone. PMID:23471564

k-Space Image Correlation Spectroscopy: A Method for Accurate Transport Measurements Independent of Fluorophore Photophysics

PubMed Central

Kolin, David L.; Ronis, David; Wiseman, Paul W.

2006-01-01

We present the theory and application of reciprocal space image correlation spectroscopy (kICS). This technique measures the number density, diffusion coefficient, and velocity of fluorescently labeled macromolecules in a cell membrane imaged on a confocal, two-photon, or total internal reflection fluorescence microscope. In contrast to r-space correlation techniques, we show kICS can recover accurate dynamics even in the presence of complex fluorophore photobleaching and/or “blinking”. Furthermore, these quantities can be calculated without nonlinear curve fitting, or any knowledge of the beam radius of the exciting laser. The number densities calculated by kICS are less sensitive to spatial inhomogeneity of the fluorophore distribution than densities measured using image correlation spectroscopy. We use simulations as a proof-of-principle to show that number densities and transport coefficients can be extracted using this technique. We present calibration measurements with fluorescent microspheres imaged on a confocal microscope, which recover Stokes-Einstein diffusion coefficients, and flow velocities that agree with single particle tracking measurements. We also show the application of kICS to measurements of the transport dynamics of α5-integrin/enhanced green fluorescent protein constructs in a transfected CHO cell imaged on a total internal reflection fluorescence microscope using charge-coupled device area detection. PMID:16861272

Spectroscopic techniques to study the immune response in human saliva

NASA Astrophysics Data System (ADS)

Nepomnyashchaya, E.; Savchenko, E.; Velichko, E.; Bogomaz, T.; Aksenov, E.

2018-01-01

Studies of the immune response dynamics by means of spectroscopic techniques, i.e., laser correlation spectroscopy and fluorescence spectroscopy, are described. The laser correlation spectroscopy is aimed at measuring sizes of particles in biological fluids. The fluorescence spectroscopy allows studying of the conformational and other structural changings in immune complex. We have developed a new scheme of a laser correlation spectrometer and an original signal processing algorithm. We have suggested a new fluorescence detection scheme based on a prism and an integrating pin diode. The developed system based on the spectroscopic techniques allows studies of complex process in human saliva and opens some prospects for an individual treatment of immune diseases.

Evidence of a glycemic threshold for the formation of pentosidine in diabetic dog lens but not in collagen.

PubMed

Nagaraj, R H; Kern, T S; Sell, D R; Fogarty, J; Engerman, R L; Monnier, V M

1996-05-01

The relationship between long-term glycemic control and the advanced Maillard reaction was investigated in dura mater collagen and lens proteins from dogs that were diabetic for 5 years. Diabetic dogs were assigned prospectively to good, moderate, and poor glycemic control and maintained by insulin. Biochemical changes were determined at study exit. Mean levels of collagen digestibility by pepsin decreased (NS) whereas collagen glycation (P < 0.001), pentosidine cross-links (P < 0.001), and collagen fluorescence (P = 0.02) increased with increasing mean HbA1 values. Similarly, mean levels of lens crystallin glycation (P < 0.001), fluorescence (P < 0.001), and the specific advanced lens Maillard product 1 (LM-1) (P < 0.001) and pentosidine (P < 0.005) increased significantly with poorer glycemic control. Statistical analysis revealed very high Spearman correlation coefficients between collagen and lens changes. Whereas pentosidine cross-links were significantly elevated in collagen from diabetic dogs with moderate levels of HbA1 (i.e., 8.0 +/- 0.4%), lens pentosidine levels were normal in this group and were elevated (P < 0.001) only in the animals with poor glycemic control (HbA1 = 9.7 +/- 0.6%). Thus, whereas protein glycation and advanced glycation in the extracellular matrix and in the lens are generally related to the level of glycemic control, there is evidence for a tissue-specific glycemic threshold for pentosidine formation, i.e., glycoxidation, in the lens. This threshold may be in part linked to a dramatic acceleration in crystallin glycation with HbA1 values of > 8.0% and/or a loss of lens membrane permeability. This study provides support at the molecular level for the growing concept that glycemic thresholds may be involved in the development of some of the complications in diabetes.

Viscosity-dependent diffusion of fluorescent particles using fluorescence correlation spectroscopy.

PubMed

Jung, Chanbae; Lee, Jaeran; Kang, Manil; Kim, Sok Won

2014-11-01

Fluorescent particles show the variety characteristics by the interaction with other particles and solvent. In order to investigate the relationship between the dynamic properties of fluorescent particles and solvent viscosity, particle diffusion in various solvents was evaluated using a fluorescence correlation spectroscopy. Upon analyzing the correlation functions of AF-647, Q-dot, and beads with different viscosity values, the diffusion time of all particles was observed to increase with increasing solvent viscosity, and the ratio of diffusion time to solvent viscosity, τ D /η, showed a linear dependence on particle size. The particle diffusion coefficients calculated from the diffusion time decreased with increasing solvent viscosity. Further, the hydrodynamic radii of AF-647, Q-dot, and beads were 0.98 ± 0.1 nm, 64.8 ± 3.23 nm, and 89.8 ± 4.91 nm, respectively, revealing a linear dependence on τ D /η, which suggests that the hydrodynamic radius of a particle strongly depends on both the physical size of the particle and solvent viscosity.

Validation of photosynthetic-fluorescence parameters as biomarkers for isoproturon toxic effect on alga Scenedesmus obliquus.

PubMed

Dewez, David; Didur, Olivier; Vincent-Héroux, Jonathan; Popovic, Radovan

2008-01-01

Photosynthetic-fluorescence parameters were investigated to be used as valid biomarkers of toxicity when alga Scenedesmus obliquus was exposed to isoproturon [3-(4-isopropylphenyl)-1,1-dimethylurea] effect. Chlorophyll fluorescence induction of algal cells treated with isoproturon showed inactivation of photosystem II (PSII) reaction centers and strong inhibition of PSII electron transport. A linear correlation was found (R2>or=0.861) between the change of cells density affected by isoproturon and the change of effective PSII quantum yield (PhiM'), photochemical quenching (qP) and relative photochemical quenching (qP(rel)) values. The cells density was also linearly dependent (R2=0.838) on the relative unquenched fluorescence parameter (UQF(rel)). Non-linear correlation was found (R2=0.937) only between cells density and the energy transfer efficiency from absorbed light to PSII reaction center (ABS/RC). The order of sensitivity determined by the EC-50% was: UQF(rel)>PhiM'>qP>qP(rel)>ABS/RC. Correlations between cells density and those photosynthetic-fluorescence parameters provide supporting evidence to use them as biomarkers of toxicity for environmental pollutants.

Time-resolved fluorescence spectroscopic study of flavin fluorescence in purified enzymes of bioluminescent bacteria

NASA Astrophysics Data System (ADS)

Vetrova, Elena; Kudryasheva, N.; Cheng, K.

2006-10-01

Time-resolved fluorescence intensity and anisotropy decay measurements have been used to study the environment and rotational mobility of endogenous flavin in two purified enzymes of bioluminescent bacteria, Luciferase from Photobacterium leiognathi and NAD(P)H:FMN-oxidoreductase from Vibrio fischeri. We compared the time-resolved fluorescence parameters, intensity decay lifetimes, rotational correlation times, and their fractional contribution, of the endogeneous flavin fluorescence in each of the two enzymes in the presence or absence of quinones of different structures and redox potentials. The endogeneous flavin exhibited multi-exponential decay characteristics as compared to a single decay lifetime of around 5 ns for free flavin, suggesting a complex and heterogeneous environment of flavin bound to the enzyme. In addition, a significant increase in the rotational correlation time and a certain degree of ordering of the molecule were observed for endogenous flavin when compared to a single and fast rotational correlation time of 150 ps of free flavin. Quinone significantly altered both the lifetime and rotational characteristics of endogenous flavin suggesting specific interactions of quinones to the endogeneous flavin in the bacterial enzyme.

Faithful reconstruction of digital holograms captured by FINCH using a Hamming window function in the Fresnel propagation.

PubMed

Siegel, Nisan; Rosen, Joseph; Brooker, Gary

2013-10-01

Recent advances in Fresnel incoherent correlation holography (FINCH) increase the signal-to-noise ratio in hologram recording by interference of images from two diffractive lenses with focal lengths close to the image plane. Holograms requiring short reconstruction distances are created that reconstruct poorly with existing Fresnel propagation methods. Here we show a dramatic improvement in reconstructed fluorescent images when a 2D Hamming window function substituted for the disk window typically used to bound the impulse response in the Fresnel propagation. Greatly improved image contrast and quality are shown for simulated and experimentally determined FINCH holograms using a 2D Hamming window without significant loss in lateral or axial resolution.

Constrained Analysis of Fluorescence Anisotropy Decay:Application to Experimental Protein Dynamics

PubMed Central

Feinstein, Efraim; Deikus, Gintaras; Rusinova, Elena; Rachofsky, Edward L.; Ross, J. B. Alexander; Laws, William R.

2003-01-01

Hydrodynamic properties as well as structural dynamics of proteins can be investigated by the well-established experimental method of fluorescence anisotropy decay. Successful use of this method depends on determination of the correct kinetic model, the extent of cross-correlation between parameters in the fitting function, and differences between the timescales of the depolarizing motions and the fluorophore's fluorescence lifetime. We have tested the utility of an independently measured steady-state anisotropy value as a constraint during data analysis to reduce parameter cross correlation and to increase the timescales over which anisotropy decay parameters can be recovered accurately for two calcium-binding proteins. Mutant rat F102W parvalbumin was used as a model system because its single tryptophan residue exhibits monoexponential fluorescence intensity and anisotropy decay kinetics. Cod parvalbumin, a protein with a single tryptophan residue that exhibits multiexponential fluorescence decay kinetics, was also examined as a more complex model. Anisotropy decays were measured for both proteins as a function of solution viscosity to vary hydrodynamic parameters. The use of the steady-state anisotropy as a constraint significantly improved the precision and accuracy of recovered parameters for both proteins, particularly for viscosities at which the protein's rotational correlation time was much longer than the fluorescence lifetime. Thus, basic hydrodynamic properties of larger biomolecules can now be determined with more precision and accuracy by fluorescence anisotropy decay. PMID:12524313

[Flag leaf photosynthetic characteristics, change in chlorophyll fluorescence parameters, and their relationships with yield of winter wheat sowed in spring].

PubMed

Xu, Lan; Gao, Zhi-qang; An, Wei; Li, Yan-liang; Jiao, Xiong-fei; Wang, Chuang-yun

2016-01-01

With five good winter wheat cultivars selected from the middle and lower reaches of Yangtze River and Southwest China as test materials, a field experiment in Xinding basin area of Shanxi Province was conducted to study the photosynthetic characteristics, chlorophyll content, and chlorophyll fluorescence parameters of flag leaf at different sowing dates, as well as the correlations between these indices and yield for two years (2013-2014). The results showed that the difference in most fluorescence parameters except chlorophyll content among cultivars was significant. The correlations between these fluorescence parameters and yield were significant. The variation coefficient of chlorophyll (Chl) content was low (0.12-0.17), and that of performance index based on absorption (PIabs) was high (0.32-0.39), with the partial correlation coefficients of them with grain yield from 2013 to 2014 ranged in 0.70-0.81. Under the early sowing condition, the grain yield positively correlated with PIabs at flowering and filling stages and chlorophyll content at grain filling stage, but negatively correlated with the relative variable fluorescence at I point (Vi) at grain filling stage. About 81.1%-82.8% of grain yield were determined by the variations of PIabs, Chl, and Vi. Wheat cultivars had various performances in the treatments with different sowing dates and a consistent trend was observed in the two experimental years. Among these 5 cultivars, Yangmai 13 was suitable for early sowing, with the flag leaf photosynthetic rate (Pn), Chl, most fluorescence parame-ters, and grain yield showed obviously high levels. In conclusion, under early sowing condition chlorophyll content at grain filling stages, PIabs at flowering and filling stages, and Pn were important indices for selecting wheat cultivars with high photosynthetic efficiency.

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.

qF-SSOP: real-time optical property corrected fluorescence imaging

PubMed Central

Valdes, Pablo A.; Angelo, Joseph P.; Choi, Hak Soo; Gioux, Sylvain

2017-01-01

Fluorescence imaging is well suited to provide image guidance during resections in oncologic and vascular surgery. However, the distorting effects of tissue optical properties on the emitted fluorescence are poorly compensated for on even the most advanced fluorescence image guidance systems, leading to subjective and inaccurate estimates of tissue fluorophore concentrations. Here we present a novel fluorescence imaging technique that performs real-time (i.e., video rate) optical property corrected fluorescence imaging. We perform full field of view simultaneous imaging of tissue optical properties using Single Snapshot of Optical Properties (SSOP) and fluorescence detection. The estimated optical properties are used to correct the emitted fluorescence with a quantitative fluorescence model to provide quantitative fluorescence-Single Snapshot of Optical Properties (qF-SSOP) images with less than 5% error. The technique is rigorous, fast, and quantitative, enabling ease of integration into the surgical workflow with the potential to improve molecular guidance intraoperatively. PMID:28856038

Fluorescent Lamp Replacement Study

DTIC Science & Technology

2017-07-01

friendly products, advances in efficiency, and lower production costs for lamps. The conversion of fluorescent bulbs to LED technology has many benefits ...of 4727 W. An economic analysis was calculated to compare the various lighting technologies that were implemented at ATC and the cost benefits ...the various lighting technologies that were implemented at ATC and the cost benefits of each, a lifecycle comparison was made between the fluorescent

Correlative cryogenic tomography of cells using light and soft x-rays.

PubMed

Smith, Elizabeth A; Cinquin, Bertrand P; Do, Myan; McDermott, Gerry; Le Gros, Mark A; Larabell, Carolyn A

2014-08-01

Correlated imaging is the process of imaging a specimen with two complementary modalities, and then combining the two data sets to create a highly informative, composite view. A recent implementation of this concept has been the combination of soft x-ray tomography (SXT) with fluorescence cryogenic microscopy (FCM). SXT-FCM is used to visualize cells that are held in a near-native, cryopreserved. The resultant images are, therefore, highly representative of both the cellular architecture and molecular organization in vivo. SXT quantitatively visualizes the cell and sub-cellular structures; FCM images the spatial distribution of fluorescently labeled molecules. Here, we review the characteristics of SXT-FCM, and briefly discuss how this method compares with existing correlative imaging techniques. We also describe how the incorporation of a cryo-rotation stage into a cryogenic fluorescence microscope allows acquisition of fluorescence cryogenic tomography (FCT) data. FCT is optimally suited for correlation with SXT, since both techniques image the specimen in 3-D, potentially with similar, isotropic spatial resolution. © 2013 Elsevier B.V. All rights reserved.

Advanced lighting guidelines: 1993. Final report

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

Eley, C.; Tolen, T.M.; Benya, J.R.

1993-12-31

The 1993 Advanced Lighting Guidelines document consists of twelve guidelines that provide an overview of specific lighting technologies and design application techniques utilizing energy-efficient lighting practice. Lighting Design Practice assesses energy-efficient lighting strategies, discusses lighting issues, and explains how to obtain quality lighting design and consulting services. Luminaires and Lighting Systems surveys luminaire equipment designed to take advantage of advanced technology lamp products and includes performance tables that allow for accurate estimation of luminaire light output and power input. The additional ten guidelines -- Computer-Aided Lighting Design, Energy-Efficient Fluorescent Ballasts, Full-Size Fluorescent Lamps, Compact Fluorescent Lamps, Tungsten-Halogen Lamps, Metal Halidemore » and HPS Lamps, Daylighting and Lumen Maintenance, Occupant Sensors, Time Scheduling Systems, and Retrofit Control Technologies -- each provide a product technology overview, discuss current products on the lighting equipment market, and provide application techniques. This document is intended for use by electric utility personnel involved in lighting programs, lighting designers, electrical engineers, architects, lighting manufacturers` representatives, and other lighting professionals.« less

Entangled-photon coincidence fluorescence imaging

PubMed Central

Scarcelli, Giuliano; Yun, Seok H.

2009-01-01

We describe fluorescence imaging using the second-order correlation of entangled photon pairs. The proposed method is based on the principle that one photon of the pair carries information on where the other photon has been absorbed and has produced fluorescence in a sample. Because fluorescent molecules serve as “detectors” breaking the entanglement, multiply-scattered fluorescence photons within the sample do not cause image blur. We discuss experimental implementations. PMID:18825257

Production of Superoxide in Bacteria Is Stress- and Cell State-Dependent: A Gating-Optimized Flow Cytometry Method that Minimizes ROS Measurement Artifacts with Fluorescent Dyes.

PubMed

McBee, Megan E; Chionh, Yok H; Sharaf, Mariam L; Ho, Peiying; Cai, Maggie W L; Dedon, Peter C

2017-01-01

The role of reactive oxygen species (ROS) in microbial metabolism and stress response has emerged as a major theme in microbiology and infectious disease. Reactive fluorescent dyes have the potential to advance the study of ROS in the complex intracellular environment, especially for high-content and high-throughput analyses. However, current dye-based approaches to measuring intracellular ROS have the potential for significant artifacts. Here, we describe a robust platform for flow cytometric quantification of ROS in bacteria using fluorescent dyes, with ROS measurements in 10s-of-1000s of individual cells under a variety of conditions. False positives and variability among sample types (e.g., bacterial species, stress conditions) are reduced with a flexible four-step gating scheme that accounts for side- and forward-scattered light (morphological changes), background fluorescence, DNA content, and dye uptake to identify cells producing ROS. Using CellROX Green dye with Escherichia coli, Mycobacterium smegmatis , and Mycobacterium bovis BCG as diverse model bacteria, we show that (1) the generation of a quantifiable CellROX Green signal for superoxide, but not hydrogen peroxide-induced hydroxyl radicals, validates this dye as a superoxide detector; (2) the level of dye-detectable superoxide does not correlate with cytotoxicity or antibiotic sensitivity; (3) the non-replicating, antibiotic tolerant state of nutrient-deprived mycobacteria is associated with high levels of superoxide; and (4) antibiotic-induced production of superoxide is idiosyncratic with regard to both the species and the physiological state of the bacteria. We also show that the gating method is applicable to other fluorescent indicator dyes, such as the 5-carboxyfluorescein diacetate acetoxymethyl ester and 5-cyano-2,3-ditolyl tetrazolium chloride for cellular esterase and reductive respiratory activities, respectively. These results demonstrate that properly controlled flow cytometry coupled with fluorescent probes provides precise and accurate quantitative analysis of ROS generation and metabolic changes in stressed bacteria.

Evanescent wave fluorescence biosensors: Advances of the last decade

PubMed Central

Taitt, Chris Rowe; Anderson, George P.; Ligler, Frances S.

2015-01-01

Biosensor development has been a highly dynamic field of research and has progressed rapidly over the past two decades. The advances have accompanied the breakthroughs in molecular biology, nanomaterial sciences, and most importantly computers and electronics. The subfield of evanescent wave fluorescence biosensors has also matured dramatically during this time. Fundamentally, this review builds on our earlier 2005 review. While a brief mention of seminal early work will be included, this current review will focus on new technological developments as well as technology commercialized in just the last decade. Evanescent wave biosensors have found a wide array applications ranging from clinical diagnostics to biodefense to food testing; advances in those applications and more are described herein. PMID:26232145

Ongoing advances in quantitative PpIX fluorescence guided intracranial tumor resection (Conference Presentation)

NASA Astrophysics Data System (ADS)

Olson, Jonathan D.; Kanick, Stephen C.; Bravo, Jaime J.; Roberts, David W.; Paulsen, Keith D.

2016-03-01

Aminolevulinc-acid induced protoporphyrin IX (ALA-PpIX) is being investigated as a biomarker to guide neurosurgical resection of brain tumors. ALA-PpIX fluorescence can be observed visually in the surgical field; however, raw fluorescence emissions can be distorted by factors other than the fluorophore concentration. Specifically, fluorescence emissions are mixed with autofluorescence and attenuated by background absorption and scattering properties of the tissue. Recent work at Dartmouth has developed advanced fluorescence detection approaches that return quantitative assessments of PpIX concentration, which are independent of background optical properties. The quantitative fluorescence imaging (qFI) approach has increased sensitivity to residual disease within the resection cavity at the end of surgery that was not visible to the naked eye through the operating microscope. This presentation outlines clinical observations made during an ongoing investigation of ALA-PpIX based guidance of tumor resection. PpIX fluorescence measurements made in a wide-field hyperspectral imaging approach are co-registered with point-assessment using a fiber optic probe. Data show variations in the measured PpIX accumulation among different clinical tumor grades (i.e. high grade glioma, low grade glioma), types (i.e. primary tumors. metastases) and normal structures of interest (e.g. normal cortex, hippocampus). These results highlight the contrast enhancement and underscore the potential clinical benefit offered from quantitative measurements of PpIX concentration during resection of intracranial tumors.

A Study of Aberrant Glycosylation in Simulated Microgravity Using Laser Induced AutoFluorescence and Flow Cytometry

NASA Technical Reports Server (NTRS)

Lawless, B. DeSales

1999-01-01

A number of pathologies and cellular dysfunctions including neoplasms have been correlated with autofluorescence. The complications of aging and diabetes have been associated with the accumulation of non-enzymatic glycosylations of tissue macromolecules. These products are known as the Advanced Glycosylated End Products (AGEs). A physical property associated with AGEs is the emission of 570 mn or 630 nm light energy (autofluorescence) following the absorption of 448 mm energy associated with the argon laser. This investigation sought to assess the induction of argon-laser induced autofluorescence in a variety of in vitro culture systems. Different fluorescence intensities distinguished tumor lines from normal cell populations. Laser-stimulated autofluorescence discriminated primary cultures of lymphocytes grown in the presence of excess glucose as opposed to normal glucose concentrations. The effects of deglycosylating agents upon laser-induced autofluorescence were also assessed. The studies included studies of cell cycle analysis using Propidium Iodide stained DNA of cells grown in simulated microgravity using NASA Bioreactor Vessels in media of normal and elevated glucose concentrations.

High-Speed PLIF Imaging of Hypersonic Transition over Discrete Cylindrical Roughness

NASA Technical Reports Server (NTRS)

Danehy, P. M.; Ivey, C. B.; Inman, J. A.; Bathel, B. F.; Jones, S. B.; McCrea, A. C.; Jiang, N.; Webster, M.; Lempert, W.; Miller, J.;

Pancreatic tissue assessment using fluorescence and reflectance spectroscopy

NASA Astrophysics Data System (ADS)

Chandra, Malavika; Heidt, David; Simeone, Diane; McKenna, Barbara; Scheiman, James; Mycek, Mary-Ann

2007-07-01

The ability of multi-modal optical spectroscopy to detect signals from pancreatic tissue was demonstrated by studying human pancreatic cancer xenografts in mice and freshly excised human pancreatic tumor tissue. Measured optical spectra and fluorescence decays were correlated with tissue morphological and biochemical properties. The measured spectral features and decay times correlated well with expected pathological differences in normal, pancreatitis and adenocarcinoma tissue states. The observed differences between the fluorescence and reflectance properties of normal, pancreatitis and adenocarcinoma tissue indicate a possible application of multi-modal optical spectroscopy to differentiating between the three tissue classifications.

Counting and behavior of an individual fluorescent molecule without hydrodynamic flow, immobilization, or photon count statistics.

PubMed

Földes-Papp, Zeno; Baumann, Gerd; Demel, Ulrike; Tilz, Gernot P

2004-04-01

Many theoretical models of molecular interactions, biochemical and chemical reactions are described on the single-molecule level, although our knowledge about the biochemical/chemical structure and dynamics primarily originates from the investigation of many-molecule systems. At present, there are four experimental platforms to observe the movement and the behavior of single fluorescent molecules: wide-field epi-illumination, near-field optical scanning, and laser scanning confocal and multiphoton microscopy. The platforms are combined with analytical methods such as fluorescence resonance energy transfer (FRET), fluorescence auto-or two-color cross-correlation spectroscopy (FCS), fluorescence polarizing anisotropy, fluorescence quenching and fluorescence lifetime measurements. The original contribution focuses on counting and characterization of freely diffusing single molecules in a single-phase like a solution or a membrane without hydrodynamic flow, immobilization or burst size analysis of intensity traces. This can be achieved, for example, by Fluorescence auto- or two-color cross-Correlation Spectroscopy as demonstrated in this original article. Three criteria (Földes-Papp (2002) Pteridines, 13, 73-82; Földes-Papp et al. (2004a) J. Immunol. Meth., 286, 1-11; Földes-Papp et al. (2004b) J. Immunol. Meth., 286, 13-20) are discussed for performing continuous measurements with one and the same single (individual) molecule, freely diffusing in a solution or a membrane, from sub-milliseconds up to severals hours. The 'algorithms' developed for single-molecule fluorescence detection are called the 'selfsame single-fluorescent-molecule regime'. An interesting application of the results found is in the field of immunology. The application of the theory to experimental results shows that the theory is consistent with the experiments. The exposition of the novel ideas on Single (Solution)-Phase Single-Molecule Fluorescence auto- or two-color cross-Correlation Spectroscopy (SPSM-FCS) are comprehensively presented. As technology continues to improve, the limits of what FCS/FCCS is being asked to do are concomitantly pushed.

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

A correlative study of Papanicolaou smear, fluorescent antibody, and culture for the diagnosis of Chlamydia trachomatis.

PubMed

Spence, M R; Barbacci, M; Kappus, E; Quinn, T

1986-11-01

A prospective study of 300 patients undergoing therapeutic termination of pregnancy was conducted. A Papanicolaou smear was obtained and a clinical evaluation of the cervix was made. Specimens from the cervix were examined by both direct fluorescent antibody and culture techniques for the presence of Chlamydia trachomatis. The presence of inflammation on Papanicolaou smear could be correlated with C trachomatis isolation. Papanicolaou smear findings consistent with C trachomatis lacked both sensitivity and specificity when compared with direct fluorescent antibody and/or culture techniques. A correlation was found between the clinical diagnosis of cervicitis and C trachomatis. This interrelationship was absent when the component findings of cervicitis (ectopy, friability, and purulent mucus) were examined independently.

Palus Somni - Anomalies in the correlation of Al/Si X-ray fluorescence intensity ratios and broad-spectrum visible albedos. [lunar surface mineralogy

NASA Technical Reports Server (NTRS)

Clark, P. E.; Andre, C. G.; Adler, I.; Weidner, J.; Podwysocki, M.

1976-01-01

The positive correlation between Al/Si X-ray fluorescence intensity ratios determined during the Apollo 15 lunar mission and a broad-spectrum visible albedo of the moon is quantitatively established. Linear regression analysis performed on 246 1 degree geographic cells of X-ray fluorescence intensity and visible albedo data points produced a statistically significant correlation coefficient of .78. Three distinct distributions of data were identified as (1) within one standard deviation of the regression line, (2) greater than one standard deviation below the line, and (3) greater than one standard deviation above the line. The latter two distributions of data were found to occupy distinct geographic areas in the Palus Somni region.

Fluorescence Behavior and Dural Infiltration of Meningioma Analyzed by 5-Aminolevulinic Acid-Based Fluorescence: Operating Microscope Versus Mini-Spectrometer.

PubMed

Knipps, Johannes; Beseoglu, Kerim; Kamp, Marcel; Fischer, Igor; Felsberg, Joerg; Neumann, Lisa M; Steiger, Hans-Jakob; Cornelius, Jan F

2017-12-01

To compare fluorescence intensity of tumor specimens, as measured by a fluorescence-guided surgery microscope and a spectrometer, to evaluate tumor infiltration of dura mater around meningiomas with help of these 2 different 5-aminolevulinic acid (5-ALA)-based fluorescence tools, and to correlate fluorescence intensity with histopathologic data. In a clinical series, meningiomas were resected by 5-ALA fluorescence-guided surgery. Fluorescence intensity was semiquantitatively rated by the surgeon at predefined points. Biopsies were harvested and fluorescence intensity measured by a spectrometer and histopathologically analyzed. Sampling was realized at the level of the dura in a centrifugal direction. A total of 104 biopsies (n = 13 tumors) were analyzed. Specificity and sensitivity of the microscope were 0.96 and 0.53 and of the spectrometer 0.95 and 0.93, respectively. Fluorescence intensity as measured by the spectrometer was correlated to histologically confirmed tumor burden. In a centrifugal direction, tumor burden and fluorescence intensity continuously decreased (along the dural tail). Below a threshold value of 639 arbitrary units no tumor was histologically detectable. At the level of the dura the spectrometer was highly sensitive for detection of meningioma cells. The surgical microscope showed false negative results and missed residual tumor cells in more than one half of the cases. The complementary use of both fluorescence tools may improve resection quality. Copyright © 2017 Elsevier Inc. All rights reserved.

Two-dimensional fluorescence lifetime correlation spectroscopy. 2. Application.

PubMed

Ishii, Kunihiko; Tahara, Tahei

2013-10-03

In the preceding article, we introduced the theoretical framework of two-dimensional fluorescence lifetime correlation spectroscopy (2D FLCS). In this article, we report the experimental implementation of 2D FLCS. In this method, two-dimensional emission-delay correlation maps are constructed from the photon data obtained with the time-correlated single photon counting (TCSPC), and then they are converted to 2D lifetime correlation maps by the inverse Laplace transform. We develop a numerical method to realize reliable transformation, employing the maximum entropy method (MEM). We apply the developed actual 2D FLCS to two real systems, a dye mixture and a DNA hairpin. For the dye mixture, we show that 2D FLCS is experimentally feasible and that it can identify different species in an inhomogeneous sample without any prior knowledge. The application to the DNA hairpin demonstrates that 2D FLCS can disclose microsecond spontaneous dynamics of biological molecules in a visually comprehensible manner, through identifying species as unique lifetime distributions. A FRET pair is attached to the both ends of the DNA hairpin, and the different structures of the DNA hairpin are distinguished as different fluorescence lifetimes in 2D FLCS. By constructing the 2D correlation maps of the fluorescence lifetime of the FRET donor, the equilibrium dynamics between the open and the closed forms of the DNA hairpin is clearly observed as the appearance of the cross peaks between the corresponding fluorescence lifetimes. This equilibrium dynamics of the DNA hairpin is clearly separated from the acceptor-missing DNA that appears as an isolated diagonal peak in the 2D maps. The present study clearly shows that newly developed 2D FLCS can disclose spontaneous structural dynamics of biological molecules with microsecond time resolution.

Characterizing harmful advanced glycation end-products (AGEs) and ribosylated aggregates of yellow mustard seed phytocystatin: Effects of different monosaccharides

NASA Astrophysics Data System (ADS)

Ahmed, Azaj; Shamsi, Anas; Bano, Bilqees

2017-01-01

Advanced glycation end products (AGEs) are at the core of variety of diseases ranging from diabetes to renal failure and hence gaining wide consideration. This study was aimed at characterizing the AGEs of phytocystatin isolated from mustard seeds (YMP) when incubated with different monosaccharides (glucose, ribose and mannose) using fluorescence, ultraviolet, circular dichroism (CD) spectroscopy and microscopy. Ribose was found to be the most potent glycating agent as evident by AGEs specific fluorescence and absorbance. YMP exists as a molten globule like structure on day 24 as depicted by high ANS fluorescence and altered intrinsic fluorescence. Glycated YMP as AGEs and ribose induced aggregates were observed at day 28 and 32 respectively. In our study we have also examined the anti-aggregative potential of polyphenol, resveratrol. Our results suggested the anti-aggregative behavior of resveratrol as it prevented the in vitro aggregation of YMP, although further studies are required to decode the mechanism by which resveratrol prevents the aggregation.

Light-sheet fluorescence imaging to localize cardiac lineage and protein distribution

PubMed Central

Ding, Yichen; Lee, Juhyun; Ma, Jianguo; Sung, Kevin; Yokota, Tomohiro; Singh, Neha; Dooraghi, Mojdeh; Abiri, Parinaz; Wang, Yibin; Kulkarni, Rajan P.; Nakano, Atsushi; Nguyen, Thao P.; Fei, Peng; Hsiai, Tzung K.

2017-01-01

Light-sheet fluorescence microscopy (LSFM) serves to advance developmental research and regenerative medicine. Coupled with the paralleled advances in fluorescence-friendly tissue clearing technique, our cardiac LSFM enables dual-sided illumination to rapidly uncover the architecture of murine hearts over 10 by 10 by 10 mm3 in volume; thereby allowing for localizing progenitor differentiation to the cardiomyocyte lineage and AAV9-mediated expression of exogenous transmembrane potassium channels with high contrast and resolution. Without the steps of stitching image columns, pivoting the light-sheet and sectioning the heart mechanically, we establish a holistic strategy for 3-dimentional reconstruction of the “digital murine heart” to assess aberrant cardiac structures as well as the spatial distribution of the cardiac lineages in neonates and ion-channels in adults. PMID:28165052

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.

Advances in Remote Sensing of Vegetation Merging NDVI, Soil Moisture, and Chlorophyll Fluorescence

NASA Astrophysics Data System (ADS)

Tucker, Compton

2016-04-01

I will describe an advance in remote sensing of vegetation in the time domain that combines simultaneous measurements of the normalized difference vegetation index, soil moisture, and chlorophyll fluorescence, all from different satellite sensors but acquired for the same areas at the same time step. The different sensor data are MODIS NDVI data from both Terra and Aqua platforms, soil moisture data from SMOS & SMP (aka SMAP but with only the passive radiometer), and chlorophyll fluorescence data from GOME-2. The complementary combination of these data provide important crop yield information for agricultural production estimates at critical phenological times in the growing season, provide a scientific basis to map land degradation, and enable quantitative determination of the end of the growing season in temperate zones.

Tracking Image Correlation: Combining Single-Particle Tracking and Image Correlation

PubMed Central

Dupont, A.; Stirnnagel, K.; Lindemann, D.; Lamb, D.C.

2013-01-01

The interactions and coordination of biomolecules are crucial for most cellular functions. The observation of protein interactions in live cells may provide a better understanding of the underlying mechanisms. After fluorescent labeling of the interacting partners and live-cell microscopy, the colocalization is generally analyzed by quantitative global methods. Recent studies have addressed questions regarding the individual colocalization of moving biomolecules, usually by using single-particle tracking (SPT) and comparing the fluorescent intensities in both color channels. Here, we introduce a new method that combines SPT and correlation methods to obtain a dynamical 3D colocalization analysis along single trajectories of dual-colored particles. After 3D tracking, the colocalization is computed at each particle’s position via the local 3D image cross correlation of the two detection channels. For every particle analyzed, the output consists of the 3D trajectory, the time-resolved 3D colocalization information, and the fluorescence intensity in both channels. In addition, the cross-correlation analysis shows the 3D relative movement of the two fluorescent labels with an accuracy of 30 nm. We apply this method to the tracking of viral fusion events in live cells and demonstrate its capacity to obtain the time-resolved colocalization status of single particles in dense and noisy environments. PMID:23746509

Fluorescent nanoparticles based on AIE fluorogens for bioimaging.

PubMed

Yan, Lulin; Zhang, Yan; Xu, Bin; Tian, Wenjing

2016-02-07

Fluorescent nanoparticles (FNPs) have recently attracted increasing attention in the biomedical field because of their unique optical properties, easy fabrication and outstanding performance in imaging. Compared with conventional molecular probes including small organic dyes and fluorescent proteins, FNPs based on aggregation-induced emission (AIE) fluorogens have shown significant advantages in tunable emission and brightness, good biocompatibility, superb photo- and physical stability, potential biodegradability and facile surface functionalization. In this review, we summarize the latest advances in the development of fluorescent nanoparticles based on AIE fluorogens including polymer nanoparticles and silica nanoparticles over the past few years, and the various biomedical applications based on these fluorescent nanoparticles are also elaborated.

Shifts in the fluorescence lifetime of EGFP during bacterial phagocytosis measured by phase-sensitive flow cytometry

NASA Astrophysics Data System (ADS)

Li, Wenyan; Houston, Kevin D.; Houston, Jessica P.

2017-01-01

Phase-sensitive flow cytometry (PSFC) is a technique in which fluorescence excited state decay times are measured as fluorescently labeled cells rapidly transit a finely focused, frequency-modulated laser beam. With PSFC the fluorescence lifetime is taken as a cytometric parameter to differentiate intracellular events that are challenging to distinguish with standard flow cytometry. For example PSFC can report changes in protein conformation, expression, interactions, and movement, as well as differences in intracellular microenvironments. This contribution focuses on the latter case by taking PSFC measurements of macrophage cells when inoculated with enhanced green fluorescent protein (EGFP)-expressing E. coli. During progressive internalization of EGFP-E. coli, fluorescence lifetimes were acquired and compared to control groups. It was hypothesized that fluorescence lifetimes would correlate well with phagocytosis because phagosomes become acidified and the average fluorescence lifetime of EGFP is known to be affected by pH. We confirmed that average EGFP lifetimes consistently decreased (3 to 2 ns) with inoculation time. The broad significance of this work is the demonstration of how high-throughput fluorescence lifetime measurements correlate well to changes that are not easily tracked by intensity-only cytometry, which is affected by heterogeneous protein expression, cell-to-cell differences in phagosome formation, and number of bacterium engulfed.

Towards native-state imaging in biological context in the electron microscope

PubMed Central

Weston, Anne E.; Armer, Hannah E. J.

2009-01-01

Modern cell biology is reliant on light and fluorescence microscopy for analysis of cells, tissues and protein localisation. However, these powerful techniques are ultimately limited in resolution by the wavelength of light. Electron microscopes offer much greater resolution due to the shorter effective wavelength of electrons, allowing direct imaging of sub-cellular architecture. The harsh environment of the electron microscope chamber and the properties of the electron beam have led to complex chemical and mechanical preparation techniques, which distance biological samples from their native state and complicate data interpretation. Here we describe recent advances in sample preparation and instrumentation, which push the boundaries of high-resolution imaging. Cryopreparation, cryoelectron microscopy and environmental scanning electron microscopy strive to image samples in near native state. Advances in correlative microscopy and markers enable high-resolution localisation of proteins. Innovation in microscope design has pushed the boundaries of resolution to atomic scale, whilst automatic acquisition of high-resolution electron microscopy data through large volumes is finally able to place ultrastructure in biological context. PMID:19916039

Correlative Fluorescence and Electron Microscopy

PubMed Central

Schirra, Randall T.; Zhang, Peijun

2014-01-01

Correlative fluorescence and electron microscopy (CFEM) is a multimodal technique that combines dynamic and localization information from fluorescence methods with ultrastructural data from electron microscopy, to give new information about how cellular components change relative to the spatiotemporal dynamics within their environment. In this review, we will discuss some of the basic techniques and tools of the trade for utilizing this attractive research method, which is becoming a very powerful tool for biology labs. The information obtained from correlative methods has proven to be invaluable in creating consensus between the two types of microscopy, extending the capability of each, and cutting the time and expense associate with using each method separately for comparative analysis. The realization of the advantages of these methods in cell biology have led to rapid improvement in the protocols and have ushered in a new generation of instruments to reach the next level of correlation – integration. PMID:25271959

Real-time measurements of airborne biologic particles using fluorescent particle counter to evaluate microbial contamination: results of a comparative study in an operating theater.

PubMed

Dai, Chunyang; Zhang, Yan; Ma, Xiaoling; Yin, Meiling; Zheng, Haiyang; Gu, Xuejun; Xie, Shaoqing; Jia, Hengmin; Zhang, Liang; Zhang, Weijun

2015-01-01

Airborne bacterial contamination poses a risk for surgical site infection, and routine surveillance of airborne bacteria is important. Traditional methods for detecting airborne bacteria are time consuming and strenuous. Measurement of biologic particle concentrations using a fluorescent particle counter is a novel method for evaluating air quality. The current study was to determine whether the number of biologic particles detected by the fluorescent particle counter can be used to indicate airborne bacterial counts in operating rooms. The study was performed in an operating theater at a university hospital in Hefei, China. The number of airborne biologic particles every minute was quantified using a fluorescent particle counter. Microbiologic air sampling was performed every 30 minutes using an Andersen air sampler (Pusong Electronic Instruments, Changzhou, China). Correlations between the 2 different methods were analyzed by Pearson correlation coefficients. A significant correlation was observed between biologic particle and bacterial counts (Pearson correlation coefficient = 0.76), and the counting results from 2 methods both increased substantially between operations, corresponding with human movements in the operating room. Fluorescent particle counters show potential as important tools for monitoring bacterial contamination in operating theatres. Copyright © 2015 Association for Professionals in Infection Control and Epidemiology, Inc. Published by Elsevier Inc. All rights reserved.

Inducing fluorescence of uranyl acetate as a dual-purpose contrast agent for correlative light-electron microscopy with nanometre precision.

PubMed

Tuijtel, Maarten W; Mulder, Aat A; Posthuma, Clara C; van der Hoeven, Barbara; Koster, Abraham J; Bárcena, Montserrat; Faas, Frank G A; Sharp, Thomas H

2017-09-05

Correlative light-electron microscopy (CLEM) combines the high spatial resolution of transmission electron microscopy (TEM) with the capability of fluorescence light microscopy (FLM) to locate rare or transient cellular events within a large field of view. CLEM is therefore a powerful technique to study cellular processes. Aligning images derived from both imaging modalities is a prerequisite to correlate the two microscopy data sets, and poor alignment can limit interpretability of the data. Here, we describe how uranyl acetate, a commonly-used contrast agent for TEM, can be induced to fluoresce brightly at cryogenic temperatures (-195 °C) and imaged by cryoFLM using standard filter sets. This dual-purpose contrast agent can be used as a general tool for CLEM, whereby the equivalent staining allows direct correlation between fluorescence and TEM images. We demonstrate the potential of this approach by performing multi-colour CLEM of cells containing equine arteritis virus proteins tagged with either green- or red-fluorescent protein, and achieve high-precision localization of virus-induced intracellular membrane modifications. Using uranyl acetate as a dual-purpose contrast agent, we achieve an image alignment precision of ~30 nm, twice as accurate as when using fiducial beads, which will be essential for combining TEM with the evolving field of super-resolution light microscopy.

LASER BIOLOGY AND MEDICINE: Application of laser fluorimetry for determining the influence of a single amino-acid substitution on the individual photophysical parameters of a fluorescent form of a fluorescent protein mRFP1

NASA Astrophysics Data System (ADS)

Banishev, A. A.; Vrzheshch, E. P.; Shirshin, E. A.

2009-03-01

Individual photophysical parameters of the chromophore of a fluorescent protein mRFP1 and its two mutants (amino-acid substitution at position 66 - mRFP1/ Q66C and mRFP1/Q66S proteins) are determined. For this purpose, apart from conventional methods of fluorimetry and spectrophotometry, nonlinear laser fluorimetry is used. It is shown that the individual extinction coefficients of the chromophore of proteins correlate (correlation coefficient above 0.9) with the volume of the substituted amino-acid residue at position 66 (similar to the positions of the absorption, fluorescence excitation and emission maxima).

Measuring the effect of a Western diet on liver tissue architecture by FLIM autofluorescence and harmonic generation microscopy

PubMed Central

Ranjit, Suman; Dvornikov, Alexander; Dobrinskikh, Evgenia; Wang, Xiaoxin; Luo, Yuhuan; Levi, Moshe; Gratton, Enrico

2017-01-01

The phasor approach to auto-fluorescence lifetime imaging was used to identify and characterize a long lifetime species (LLS) (~7.8 ns) in livers of mice fed with a Western diet. The size of the areas containing this LLS species depends on the type of diet and the size distribution shows Western diet has much larger LLS sizes. Combination of third harmonic generation images with FLIM identified the LLS species with fat droplets and the droplet size distribution was estimated. Second harmonic generation microscopy combined with phasor FLIM shows that there is an increase in fibrosis with a Western diet. A new decomposition in three components of the phasor plot shows that a Western diet is correlated with a higher fraction of free NADH, signifying more reducing condition and more glycolytic condition. Multiparametric analysis of phasor distribution shows that from the distribution of phasor points, a Western diet fed versus a low fat diet fed samples of mice livers can be separated. The phasor approach for the analysis of FLIM images of autofluorescence in liver specimens can result in discovery of new fluorescent species and then these new fluorescent species can help assess tissue architecture. Finally integrating FLIM and second and third harmonic analysis provides a measure of the advancement of fibrosis as an effect of diet. PMID:28717559

Nanoscale volume confinement and fluorescence enhancement with double nanohole aperture

PubMed Central

Regmi, Raju; Al Balushi, Ahmed A.; Rigneault, Hervé; Gordon, Reuven; Wenger, Jérôme

2015-01-01

Diffraction ultimately limits the fluorescence collected from a single molecule, and sets an upper limit to the maximum concentration to isolate a single molecule in the detection volume. To overcome these limitations, we introduce here the use of a double nanohole structure with 25 nm gap, and report enhanced detection of single fluorescent molecules in concentrated solutions exceeding 20 micromolar. The nanometer gap concentrates the light into an apex volume down to 70 zeptoliter (10−21 L), 7000-fold below the diffraction-limited confocal volume. Using fluorescence correlation spectroscopy and time-correlated photon counting, we measure fluorescence enhancement up to 100-fold, together with local density of optical states (LDOS) enhancement of 30-fold. The distinctive features of double nanoholes combining high local field enhancement, efficient background screening and relative nanofabrication simplicity offer new strategies for real time investigation of biochemical events with single molecule resolution at high concentrations. PMID:26511149

Confocal Microscopy for the Histological Fluorescence Pattern of a Recurrent Atypical Meningioma: Case Report

PubMed Central

Whitson, Wesley J.; Valdes, Pablo A.; Harris, Brent T.; Paulsen, Keith D.; Roberts, David W.

2013-01-01

Background and Importance Fluorescence-guided resection with 5-aminolevulinic acid (5-ALA), which has shown promising results in the resection of malignant gliomas, has been used for meningioma resection in an attempt to more clearly delineate the tumor margin. However, no article has investigated the fluorescence pattern of meningiomas on a histological level. Understanding the microscopic pattern of fluorescence could help assess the precision and utility of using 5-ALA for these tumors. We present the case of a recurrent atypical meningioma operated on with 5-ALA fluorescence-guided resection for delineation of tumor tissue from surrounding uninvolved dura. Clinical Presentation A 53-year-old woman presented with recurrent atypical meningioma of the falx. Prior treatment included surgical resection 6 years earlier with subsequent fractionated radiation therapy and radiosurgery for tumor progression. The patient was given 5-ALA 20 mg/kg body weight dissolved in 100 mL water 3 hours before induction of anesthesia. Intraoperative fluorescence was coregistered with preoperative imaging. Neuropathological analysis of the resected falx with confocal microscopy enabled correlation of fluorescence with the extent of tumor on a histological level. Conclusion Fluorescence guidance allowed clear intraoperative delineation of tumor tissue from adjacent, uninvolved dura. On a microscopic level, there was a very close correlation of fluorescence with tumor, but some tumor cells did not fluoresce. PMID:21389893

Analyzing Intracellular Binding and Diffusion with Continuous Fluorescence Photobleaching

PubMed Central

Wachsmuth, Malte; Weidemann, Thomas; Müller, Gabriele; Hoffmann-Rohrer, Urs W.; Knoch, Tobias A.; Waldeck, Waldemar; Langowski, Jörg

2003-01-01

Transport and binding of molecules to specific sites are necessary for the assembly and function of ordered supramolecular structures in cells. For analyzing these processes in vivo, we have developed a confocal fluorescence fluctuation microscope that allows both imaging of the spatial distribution of fluorescent molecules with confocal laser scanning microscopy and probing their mobility at specific positions in the cell with fluorescence correlation spectroscopy and continuous fluorescence photobleaching (CP). Because fluorescence correlation spectroscopy is restricted to rapidly diffusing particles and CP to slower processes, these two methods complement each other. For the analysis of binding-related contributions to mobility we have derived analytical expressions for the temporal behavior of CP curves from which the bound fraction and/or the dissociation rate or residence time at binding sites, respectively, can be obtained. In experiments, we investigated HeLa cells expressing different fluorescent proteins: Although enhanced green fluorescent protein (EGFP) shows high mobility, fusions of histone H2B with the yellow fluorescent protein are incorporated into chromatin, and these nuclei exhibit the presence of a stably bound and a freely diffusing species. Nonpermanent binding was found for mTTF-I, a transcription termination factor for RNA polymerase I, fused with EGFP. The cells show fluorescent nucleoli, and binding is transient. CP yields residence times for mTTF-I-EGFP of ∼13 s. PMID:12719264

Analyzing intracellular binding and diffusion with continuous fluorescence photobleaching.

PubMed

Wachsmuth, Malte; Weidemann, Thomas; Müller, Gabriele; Hoffmann-Rohrer, Urs W; Knoch, Tobias A; Waldeck, Waldemar; Langowski, Jörg

2003-05-01

Transport and binding of molecules to specific sites are necessary for the assembly and function of ordered supramolecular structures in cells. For analyzing these processes in vivo, we have developed a confocal fluorescence fluctuation microscope that allows both imaging of the spatial distribution of fluorescent molecules with confocal laser scanning microscopy and probing their mobility at specific positions in the cell with fluorescence correlation spectroscopy and continuous fluorescence photobleaching (CP). Because fluorescence correlation spectroscopy is restricted to rapidly diffusing particles and CP to slower processes, these two methods complement each other. For the analysis of binding-related contributions to mobility we have derived analytical expressions for the temporal behavior of CP curves from which the bound fraction and/or the dissociation rate or residence time at binding sites, respectively, can be obtained. In experiments, we investigated HeLa cells expressing different fluorescent proteins: Although enhanced green fluorescent protein (EGFP) shows high mobility, fusions of histone H2B with the yellow fluorescent protein are incorporated into chromatin, and these nuclei exhibit the presence of a stably bound and a freely diffusing species. Nonpermanent binding was found for mTTF-I, a transcription termination factor for RNA polymerase I, fused with EGFP. The cells show fluorescent nucleoli, and binding is transient. CP yields residence times for mTTF-I-EGFP of approximately 13 s.

A novel method to visually determine the intracellular pH of xenografted tumor in vivo by utilizing fluorescent protein as an indicator.

PubMed

Tanaka, Shotaro; Harada, Hiroshi; Hiraoka, Masahiro

2015-09-04

The alkalization of intracellular pH (pHin) advances together with enhancement of aerobic glycolysis within tumor cells (the Warburg effect), and that is responsible for the progression of tumor malignancy together with hypoxia and angiogenesis. But how they correlate each other during tumor growth is poorly understood, partly due to the lack of suitable imaging methods. In present study, we propose a novel method to visually determine the pHin of tumor xenograft model from fluorescent image ratios. We utilized tandemly-linked two fluorescent proteins as a pH indicator; yellow fluorescent protein (YFP, pH sensitive) as an indicator, and red fluorescent protein (RFP, pH insensitive) as a reference. This method can eliminate the influence of optical factors from tissue as well as of the diverse expression level of pH indicator in the grafted cells. In addition, that can be operated by filter-based fluorescent imagers that are generally used in small animal study. The efficacy of the pH indicator, RFP-YFP, was confirmed by studies using recombinant protein in vitro and HeLa cells expressing RFP-YFP in vivo. Furthermore, we prepared nude mice subcutaneously xenografted HeLa cells expressing RFP-YFP cells as tumor model. The image ratios (YFP/RFP) of the tumor at the day 5 after surgery clearly showed the heterogeneous distribution of diverse pHin cells in the tumor tissue. Concomitantly acquired angiography using near-infrared fluorescence (680 nm for emission) also indicated that the relative alkaline pHin cells located in the region far from tumor vessels in which tumor aerobic glycolysis would be facilitated by progression of hypoxia and nutrient starvation. Applying the present method for a multi-wavelength imaging concerning pO2 and/or nutrient starvation states in addition to pHin and angiogenesis would provide valuable information about complicated alteration of tumoral cell states during tumorigenesis. Copyright © 2015 Elsevier Inc. All rights reserved.

Tracking quasi-stationary flow of weak fluorescent signals by adaptive multi-frame correlation.

PubMed

Ji, L; Danuser, G

2005-12-01

We have developed a novel cross-correlation technique to probe quasi-stationary flow of fluorescent signals in live cells at a spatial resolution that is close to single particle tracking. By correlating image blocks between pairs of consecutive frames and integrating their correlation scores over multiple frame pairs, uncertainty in identifying a globally significant maximum in the correlation score function has been greatly reduced as compared with conventional correlation-based tracking using the signal of only two consecutive frames. This approach proves robust and very effective in analysing images with a weak, noise-perturbed signal contrast where texture characteristics cannot be matched between only a pair of frames. It can also be applied to images that lack prominent features that could be utilized for particle tracking or feature-based template matching. Furthermore, owing to the integration of correlation scores over multiple frames, the method can handle signals with substantial frame-to-frame intensity variation where conventional correlation-based tracking fails. We tested the performance of the method by tracking polymer flow in actin and microtubule cytoskeleton structures labelled at various fluorophore densities providing imagery with a broad range of signal modulation and noise. In applications to fluorescent speckle microscopy (FSM), where the fluorophore density is sufficiently low to reveal patterns of discrete fluorescent marks referred to as speckles, we combined the multi-frame correlation approach proposed above with particle tracking. This hybrid approach allowed us to follow single speckles robustly in areas of high speckle density and fast flow, where previously published FSM analysis methods were unsuccessful. Thus, we can now probe cytoskeleton polymer dynamics in living cells at an entirely new level of complexity and with unprecedented detail.

Fluorescence lifetime imaging of skin cancer

NASA Astrophysics Data System (ADS)

Patalay, Rakesh; Talbot, Clifford; Munro, Ian; Breunig, Hans Georg; König, Karsten; Alexandrov, Yuri; Warren, Sean; Neil, Mark A. A.; French, Paul M. W.; Chu, Anthony; Stamp, Gordon W.; Dunsby, Chris

2011-03-01

Fluorescence intensity imaging and fluorescence lifetime imaging microscopy (FLIM) using two photon microscopy (TPM) have been used to study tissue autofluorescence in ex vivo skin cancer samples. A commercially available system (DermaInspect®) was modified to collect fluorescence intensity and lifetimes in two spectral channels using time correlated single photon counting and depth-resolved steady state measurements of the fluorescence emission spectrum. Uniquely, image segmentation has been used to allow fluorescence lifetimes to be calculated for each cell. An analysis of lifetime values obtained from a range of pigmented and non-pigmented lesions will be presented.

A Critical and Comparative Review of Fluorescent Tools for Live-Cell Imaging.

PubMed

Specht, Elizabeth A; Braselmann, Esther; Palmer, Amy E

2017-02-10

Fluorescent tools have revolutionized our ability to probe biological dynamics, particularly at the cellular level. Fluorescent sensors have been developed on several platforms, utilizing either small-molecule dyes or fluorescent proteins, to monitor proteins, RNA, DNA, small molecules, and even cellular properties, such as pH and membrane potential. We briefly summarize the impressive history of tool development for these various applications and then discuss the most recent noteworthy developments in more detail. Particular emphasis is placed on tools suitable for single-cell analysis and especially live-cell imaging applications. Finally, we discuss prominent areas of need in future fluorescent tool development-specifically, advancing our capability to analyze and integrate the plethora of high-content data generated by fluorescence imaging.

Imaging Cellular Dynamics with Spectral Relaxation Imaging Microscopy: Distinct Spectral Dynamics in Golgi Membranes of Living Cells.

PubMed

Lajevardipour, Alireza; Chon, James W M; Chattopadhyay, Amitabha; Clayton, Andrew H A

2016-11-22

Spectral relaxation from fluorescent probes is a useful technique for determining the dynamics of condensed phases. To this end, we have developed a method based on wide-field spectral fluorescence lifetime imaging microscopy to extract spectral relaxation correlation times of fluorescent probes in living cells. We show that measurement of the phase and modulation of fluorescence from two wavelengths permit the identification and determination of excited state lifetimes and spectral relaxation correlation times at a single modulation frequency. For NBD fluorescence in glycerol/water mixtures, the spectral relaxation correlation time determined by our approach exhibited good agreement with published dielectric relaxation measurements. We applied this method to determine the spectral relaxation dynamics in membranes of living cells. Measurements of the Golgi-specific C 6 -NBD-ceramide probe in living HeLa cells revealed sub-nanosecond spectral dynamics in the intracellular Golgi membrane and slower nanosecond spectral dynamics in the extracellular plasma membrane. We interpret the distinct spectral dynamics as a result of structural plasticity of the Golgi membrane relative to more rigid plasma membranes. To the best of our knowledge, these results constitute one of the first measurements of Golgi rotational dynamics.

Fluorescence spectroscopy for endogenous porphyrins in human facial skin

NASA Astrophysics Data System (ADS)

Seo, I.; Tseng, S. H.; Cula, G. O.; Bargo, P. R.; Kollias, N.

2009-02-01

The activity of certain bacteria in skin is known to correlate to the presence of porphyrins. In particular the presence of coproporphyrin produced by P.acnes inside plugged pores has been correlated to acne vulgaris. Another porphyrin encountered in skin is protoporphyrin IX, which is produced by the body in the pathway for production of heme. In the present work, a fluorescence spectroscopy system was developed to measure the characteristic spectrum and quantify the two types of porphyrins commonly present in human facial skin. The system is comprised of a Xe lamp both for fluorescence excitation and broadband light source for diffuse reflectance measurements. A computer-controlled filter wheel enables acquisition of sequential spectra, first excited by blue light at 405 nm then followed by the broadband light source, at the same location. The diffuse reflectance spectrum was used to correct the fluorescence spectrum due to the presence of skin chromophores, such as blood and melanin. The resulting fluorescence spectra were employed for the quantification of porphyrin concentration in a population of healthy subjects. The results show great variability on the concentration of these porphyrins and further studies are being conducted to correlate them with skin conditions such as inflammation and acne vulgaris.

Sun-induced chlorophyll fluorescence, photosynthesis, and light use efficiency of a soybean field from seasonally continuous measurements

USDA-ARS?s Scientific Manuscript database

Recent development of sun-induced chlorophyll fluorescence (SIF) technology is stimulating studies to remotely approximate canopy photosynthesis (measured as gross primary production, GPP). While multiple applications have advanced the empirical relationship between GPP and SIF, mechanistic understa...

Fluorescence guided surgery and tracer-dose, fact or fiction?

PubMed

KleinJan, Gijs H; Bunschoten, Anton; van den Berg, Nynke S; Olmos, Renato A Valdès; Klop, W Martin C; Horenblas, Simon; van der Poel, Henk G; Wester, Hans-Jürgen; van Leeuwen, Fijs W B

2016-09-01

Fluorescence guidance is an upcoming methodology to improve surgical accuracy. Challenging herein is the identification of the minimum dose at which the tracer can be detected with a clinical-grade fluorescence camera. Using a hybrid tracer such as indocyanine green (ICG)-(99m)Tc-nanocolloid, it has become possible to determine the accumulation of tracer and correlate this to intraoperative fluorescence-based identification rates. In the current study, we determined the lower detection limit of tracer at which intraoperative fluorescence guidance was still feasible. Size exclusion chromatography (SEC) provided a laboratory set-up to analyze the chemical content and to simulate the migratory behavior of ICG-nanocolloid in tissue. Tracer accumulation and intraoperative fluorescence detection findings were derived from a retrospective analysis of 20 head-and-neck melanoma patients, 40 penile and 20 prostate cancer patients scheduled for sentinel node (SN) biopsy using ICG-(99m)Tc-nanocolloid. In these patients, following tracer injection, single photon emission computed tomography fused with computed tomography (SPECT/CT) was used to identify the SN(s). The percentage injected dose (% ID), the amount of ICG (in nmol), and the concentration of ICG in the SNs (in μM) was assessed for SNs detected on SPECT/CT and correlated with the intraoperative fluorescence imaging findings. SEC determined that in the hybrid tracer formulation, 41 % (standard deviation: 12 %) of ICG was present in nanocolloid-bound form. In the SNs detected using fluorescence guidance a median of 0.88 % ID was present, compared to a median of 0.25 % ID in the non-fluorescent SNs (p-value < 0.001). The % ID values could be correlated to the amount ICG in a SN (range: 0.003-10.8 nmol) and the concentration of ICG in a SN (range: 0.006-64.6 μM). The ability to provide intraoperative fluorescence guidance is dependent on the amount and concentration of the fluorescent dye accumulated in the lesion(s) of interest. Our findings indicate that intraoperative fluorescence detection with ICG is possible above a μM concentration.

Nondestructive assessment of collagen hydrogel cross-linking using time-resolved autofluorescence imaging

NASA Astrophysics Data System (ADS)

Sherlock, Benjamin E.; Harvestine, Jenna N.; Mitra, Debika; Haudenschild, Anne; Hu, Jerry; Athanasiou, Kyriacos A.; Leach, J. Kent; Marcu, Laura

2018-03-01

We investigate the use of a fiber-based, multispectral fluorescence lifetime imaging (FLIm) system to nondestructively monitor changes in mechanical properties of collagen hydrogels caused by controlled application of widely used cross-linking agents, glutaraldehyde (GTA) and ribose. Postcross-linking, fluorescence lifetime images are acquired prior to the hydrogels being processed by rheological or tensile testing to directly probe gel mechanical properties. To preserve the sterility of the ribose-treated gels, FLIm is performed inside a biosafety cabinet (BSC). A pairwise correlation analysis is used to quantify the relationship between mean hydrogel fluorescence lifetimes and the storage or Young's moduli of the gels. In the GTA study, we observe strong and specific correlations between fluorescence lifetime and the storage and Young's moduli. Similar correlations are not observed in the ribose study and we postulate a reason for this. Finally, we demonstrate the ability of FLIm to longitudinally monitor dynamic cross-link formation. The strength of the GTA correlations and deployment of our fiber-based FLIm system inside the aseptic environment of a BSC suggests that this technique may be a valuable tool for the tissue engineering community where longitudinal assessment of tissue construct maturation in vitro is highly desirable.

Multispectral fluorescence imaging for detection of bovine feces on Romaine lettuce and baby spinach leaves

USDA-ARS?s Scientific Manuscript database

Hyperspectral fluorescence imaging with ultraviolet-A excitation was used to evaluate the feasibility of two-waveband fluorescence algorithms for the detection of bovine fecal contaminants on the abaxial and adaxial surfaces of Romaine lettuce and baby spinach leaves. Correlation analysis was used t...

Correlation Between Quantitative HER-2 Protein Expression and Risk for Brain Metastases in HER-2+ Advanced Breast Cancer Patients Receiving Trastuzumab-Containing Therapy

PubMed Central

Duchnowska, Renata; Biernat, Wojciech; Szostakiewicz, Barbara; Sperinde, Jeff; Piette, Fanny; Haddad, Mojgan; Paquet, Agnes; Lie, Yolanda; Czartoryska-Arłukowicz, Bogumiła; Wysocki, Piotr; Jankowski, Tomasz; Radecka, Barbara; Foszczyńska-Kłoda, Małgorzata; Litwiniuk, Maria; Dȩbska, Sylwia; Weidler, Jodi; Huang, Weidong; Buyse, Marc; Bates, Michael

2012-01-01

Background. Patients with human epidermal growth factor receptor (HER)-2+ breast cancer are at particularly high risk for brain metastases; however, the biological basis is not fully understood. Using a novel HER-2 assay, we investigated the correlation between quantitative HER-2 expression in primary breast cancers and the time to brain metastasis (TTBM) in HER-2+ advanced breast cancer patients treated with trastuzumab. Methods. The study group included 142 consecutive patients who were administered trastuzumab-based therapy for HER-2+ metastatic breast cancer. HER-2/neu gene copy number was quantified as the HER-2/centromeric probe for chromosome 17 (CEP17) ratio by central laboratory fluorescence in situ hybridization (FISH). HER-2 protein was quantified as total HER-2 protein expression (H2T) by the HERmark® assay (Monogram Biosciences, Inc., South San Francisco, CA) in formalin-fixed, paraffin-embedded tumor samples. HER-2 variables were correlated with clinical features and TTBM was measured from the initiation of trastuzumab-containing therapy. Results. A higher H2T level (continuous variable) was correlated with shorter TTBM, whereas HER-2 amplification by FISH and a continuous HER-2/CEP17 ratio were not predictive (p = .013, .28, and .25, respectively). In the subset of patients that was centrally determined by FISH to be HER-2+, an above-the-median H2T level was significantly associated with a shorter TTBM (hazard ratio, [HR], 2.4; p = .005), whereas this was not true for the median HER-2/CEP17 ratio by FISH (p = .4). Correlation between a continuous H2T level and TTBM was confirmed on multivariate analysis (HR, 3.3; p = .024). Conclusions. These data reveal a strong relationship between the quantitative HER-2 protein expression level and the risk for brain relapse in HER-2+ advanced breast cancer patients. Consequently, quantitative assessment of HER-2 protein expression may inform and facilitate refinements in therapeutic treatment strategies for selected subpopulations of patients in this group. PMID:22234627

Correlation between quantitative HER-2 protein expression and risk for brain metastases in HER-2+ advanced breast cancer patients receiving trastuzumab-containing therapy.

PubMed

Duchnowska, Renata; Biernat, Wojciech; Szostakiewicz, Barbara; Sperinde, Jeff; Piette, Fanny; Haddad, Mojgan; Paquet, Agnes; Lie, Yolanda; Czartoryska-Arłukowicz, Bogumiła; Wysocki, Piotr; Jankowski, Tomasz; Radecka, Barbara; Foszczynska-Kłoda, Małgorzata; Litwiniuk, Maria; Debska, Sylwia; Weidler, Jodi; Huang, Weidong; Buyse, Marc; Bates, Michael; Jassem, Jacek

2012-01-01

Patients with human epidermal growth factor receptor (HER)-2+ breast cancer are at particularly high risk for brain metastases; however, the biological basis is not fully understood. Using a novel HER-2 assay, we investigated the correlation between quantitative HER-2 expression in primary breast cancers and the time to brain metastasis (TTBM) in HER-2+ advanced breast cancer patients treated with trastuzumab. The study group included 142 consecutive patients who were administered trastuzumab-based therapy for HER-2+ metastatic breast cancer. HER-2/neu gene copy number was quantified as the HER-2/centromeric probe for chromosome 17 (CEP17) ratio by central laboratory fluorescence in situ hybridization (FISH). HER-2 protein was quantified as total HER-2 protein expression (H2T) by the HERmark® assay (Monogram Biosciences, Inc., South San Francisco, CA) in formalin-fixed, paraffin-embedded tumor samples. HER-2 variables were correlated with clinical features and TTBM was measured from the initiation of trastuzumab-containing therapy. A higher H2T level (continuous variable) was correlated with shorter TTBM, whereas HER-2 amplification by FISH and a continuous HER-2/CEP17 ratio were not predictive (p = .013, .28, and .25, respectively). In the subset of patients that was centrally determined by FISH to be HER-2+, an above-the-median H2T level was significantly associated with a shorter TTBM (hazard ratio, [HR], 2.4; p = .005), whereas this was not true for the median HER-2/CEP17 ratio by FISH (p = .4). Correlation between a continuous H2T level and TTBM was confirmed on multivariate analysis (HR, 3.3; p = .024). These data reveal a strong relationship between the quantitative HER-2 protein expression level and the risk for brain relapse in HER-2+ advanced breast cancer patients. Consequently, quantitative assessment of HER-2 protein expression may inform and facilitate refinements in therapeutic treatment strategies for selected subpopulations of patients in this group.

Integrated fluorescence correlation spectroscopy device for point-of-care clinical applications

PubMed Central

Olson, Eben; Torres, Richard; Levene, Michael J.

2013-01-01

We describe an optical system which reduces the cost and complexity of fluorescence correlation spectroscopy (FCS), intended to increase the suitability of the technique for clinical use. Integration of the focusing optics and sample chamber into a plastic component produces a design which is simple to align and operate. We validate the system by measurements on fluorescent dye, and compare the results to a commercial instrument. In addition, we demonstrate its application to measurements of concentration and multimerization of the clinically relevant protein von Willebrand factor (vWF) in human plasma. PMID:23847733

The use of transgenic parasites in malaria vaccine research.

PubMed

Othman, Ahmad Syibli; Marin-Mogollon, Catherin; Salman, Ahmed M; Franke-Fayard, Blandine M; Janse, Chris J; Khan, Shahid M

2017-07-01

Transgenic malaria parasites expressing foreign genes, for example fluorescent and luminescent proteins, are used extensively to interrogate parasite biology and host-parasite interactions associated with malaria pathology. Increasingly transgenic parasites are also exploited to advance malaria vaccine development. Areas covered: We review how transgenic malaria parasites are used, in vitro and in vivo, to determine protective efficacy of different antigens and vaccination strategies and to determine immunological correlates of protection. We describe how chimeric rodent parasites expressing P. falciparum or P. vivax antigens are being used to directly evaluate and rank order human malaria vaccines before their advancement to clinical testing. In addition, we describe how transgenic human and rodent parasites are used to develop and evaluate live (genetically) attenuated vaccines. Expert commentary: Transgenic rodent and human malaria parasites are being used to both identify vaccine candidate antigens and to evaluate both sub-unit and whole organism vaccines before they are advanced into clinical testing. Transgenic parasites combined with in vivo pre-clinical testing models (e.g. mice) are used to evaluate vaccine safety, potency and the durability of protection as well as to uncover critical protective immune responses and to refine vaccination strategies.

Correlated expression of gfp and Bt cry1Ac gene facilitates quantification of transgenic hybridization between Brassicas.

PubMed

Shen, B-C; Stewart, C N; Zhang, M-Q; Le, Y-T; Tang, Z-X; Mi, X-C; Wei, W; Ma, K-P

2006-09-01

Gene flow from transgenic oilseed rape (BRASSICA NAPUS) might not be avoidable, thus, it is important to detect and quantify hybridization events with its relatives in real time. Data are presented showing the correlation between genetically linked green fluorescent protein (GFP) with BACILLUS THURINGIENSIS (Bt) CRY1AC gene expression in hybrids formed between transgenic B. NAPUS "Westar" and a wild Chinese accession of wild mustard (B. JUNCEA) and hybridization between transgenic B. NAPUS and a conspecific Chinese landrace oilseed rape. Hybrids were obtained either by spontaneous hybridization in the field or by hand-crossing in a greenhouse. In all cases, transgenic hybrids were selected by GFP fluorescence among seedlings originating from seeds harvested from B. JUNCEA and the Chinese oilseed rape plants. Transgenicity was confirmed by PCR detection of transgenes. GFP fluorescence was easily and rapidly detected in the hybrids under greenhouse and field conditions. Results showed that both GFP fluorescence and Bt protein synthesis decreased as either plant or leaf aged, and GFP fluorescence intensity was closely correlated with Bt protein concentration during the entire vegetative lifetime in hybrids. These findings allow the use of GFP fluorescence as an accurate tool to detect gene-flow in time in the field and to conveniently estimate BT CRY1AC expression in hybrids on-the-plant.

Surface Transient Binding-Based Fluorescence Correlation Spectroscopy (STB-FCS), a Simple and Easy-to-Implement Method to Extend the Upper Limit of the Time Window to Seconds.

PubMed

Peng, Sijia; Wang, Wenjuan; Chen, Chunlai

2018-05-10

Fluorescence correlation spectroscopy is a powerful single-molecule tool that is able to capture kinetic processes occurring at the nanosecond time scale. However, the upper limit of its time window is restricted by the dwell time of the molecule of interest in the confocal detection volume, which is usually around submilliseconds for a freely diffusing biomolecule. Here, we present a simple and easy-to-implement method, named surface transient binding-based fluorescence correlation spectroscopy (STB-FCS), which extends the upper limit of the time window to seconds. We further demonstrated that STB-FCS enables capture of both intramolecular and intermolecular kinetic processes whose time scales cross several orders of magnitude.

Novel insights into anoxic/aerobic(1)/aerobic(2) biological fluidized-bed system for coke wastewater treatment by fluorescence excitation-emission matrix spectra coupled with parallel factor analysis.

PubMed

Ou, Hua-Se; Wei, Chao-Hai; Mo, Ce-Hui; Wu, Hai-Zhen; Ren, Yuan; Feng, Chun-Hua

2014-10-01

Fluorescence spectroscopy coupled with parallel factor analysis (PARAFAC) was applied to investigate the contaminant removal efficiency and fluorescent characteristic variations in a full scale coke wastewater (CWW) treatment plant with a novel anoxic/aerobic(1)/aerobic(2) (A/O(1)/O(2)) process, which combined with internal-loop fluidized-bed reactor. Routine monitoring results indicated that primary contaminants in CWW, such as phenols and free cyanide, were removed efficiently in A/O(1)/O(2) process (removal efficiency reached 99% and 95%, respectively). Three-dimensional excitation-emission matrix fluorescence spectroscopy and PARAFAC identified three fluorescent components, including two humic-like fluorescence components (C1 and C3) and one protein-like component (C2). Principal component analysis revealed that C1 and C2 correlated with COD (correlation coefficient (r)=0.782, p<0.01 and r=0.921, p<0.01), respectively) and phenols (r=0.796, p<0.01 and r=0.914, p<0.01, respectively), suggesting that C1 and C2 might be associated with the predominating aromatic contaminants in CWW. C3 correlated with mixed liquor suspended solids (r=0.863, p<0.01) in fluidized-bed reactors, suggesting that it might represent the biological dissolved organic matter. In A/O(1)/O(2) process, the fluorescence intensities of C1 and C2 consecutively decreased, indicating the degradation of aromatic contaminants. Correspondingly, the fluorescence intensity of C3 increased in aerobic(1) stage, suggesting an increase of biological dissolved organic matter. Copyright © 2014 Elsevier Ltd. All rights reserved.

Evaluation of resin infiltration using quantitative light-induced fluorescence technology.

PubMed

Min, Ji-Hyun; Inaba, Daisuke; Kim, Baek-Il

2016-09-01

To determine whether quantitative light-induced fluorescence (QLF) technology can be used to classify the colour of teeth specimens before and after resin infiltration (RI) treatment, and calculate the correlation between the ΔF value and colour difference (ΔE) in fluorescence images of the specimens obtained using a QLF-digital (QLF-D) device. Sixty sound bovine permanent teeth specimens were immersed in demineralized solution. Two exposed windows were formed in each specimen, and RI treatment was applied to one of them. The ΔE values were obtained for the differences between a sound tooth surface (SS), an early dental caries surface (ECS) and an ECS treated with RI (RS) in white-light and fluorescence images obtained using QLF-D, respectively. The ΔF value was obtained from fluorescence images using dedicated software for QLF-D. The mean differences between the ΔE values obtained from the white-light and fluorescence images were analyzed by paired t-test. Pearson correlation analysis and Bland-Altman plots were applied to the differences between the ΔF value for ECS (ΔFSS-ECS) and the ΔE value between SS and ECS (ΔESS-ECS), and between the ΔF value for RS (ΔFSS-RS) and the ΔE value between SS and RS (ΔESS-RS) in fluorescence images. The ΔE values obtained from fluorescence images were three times higher than the ΔE values obtained from white-light images (p<0.001). Significant correlations were confirmed between ΔESS-ECS and ΔFSS-ECS (r=-0.492, p<0.001) and between ΔESS-RS and ΔFSS-RS (r=-0.661, p<0.001). QLF technology can be used to confirm the presence of RI in teeth. Copyright © 2016 Elsevier B.V. All rights reserved.

Multi-Channel Hyperspectral Fluorescence Detection Excited by Coupled Plasmon-Waveguide Resonance

PubMed Central

Du, Chan; Liu, Le; Zhang, Lin; Guo, Jun; Guo, Jihua; Ma, Hui; He, Yonghong

2013-01-01

We propose in this paper a biosensor scheme based on coupled plasmon-waveguide resonance (CPWR) excited fluorescence spectroscopy. A symmetrical structure that offers higher surface electric field strengths, longer surface propagation lengths and depths is developed to support guided waveguide modes for the efficient excitation of fluorescence. The optimal parameters for the sensor films are theoretically and experimentally investigated, leading to a detection limit of 0.1 nM (for a Cy5 solution). Multiplex analysis possible with the fluorescence detection is further advanced by employing the hyperspectral fluorescence technique to record the full spectra for every pixel on the sample plane. We demonstrate experimentally that highly overlapping fluorescence (Cy5 and Dylight680) can be distinguished and ratios of different emission sources can be determined accurately. This biosensor shows great potential for multiplex detections of fluorescence analytes. PMID:24129023

Oceanographic applications of laser technology

NASA Technical Reports Server (NTRS)

Hoge, F. E.

1988-01-01

Oceanographic activities with the Airborne Oceanographic Lidar (AOL) for the past several years have primarily been focussed on using active (laser induced pigment fluorescence) and concurrent passive ocean color spectra to improve existing ocean color algorithms for estimating primary production in the world's oceans. The most significant results were the development of a technique for selecting optimal passive wavelengths for recovering phytoplankton photopigment concentration and the application of this technique, termed active-passive correlation spectroscopy (APCS), to various forms of passive ocean color algorithms. Included in this activity is use of airborne laser and passive ocean color for development of advanced satellite ocean color sensors. Promising on-wavelength subsurface scattering layer measurements were recently obtained. A partial summary of these results are shown.

Fluorescence Microscopy Gets Faster and Clearer: Roles of Photochemistry and Selective Illumination

PubMed Central

Wolenski, Joseph S.; Julich, Doerthe

2014-01-01

Significant advances in fluorescence microscopy tend be a balance between two competing qualities wherein improvements in resolution and low light detection are typically accompanied by losses in acquisition rate and signal-to-noise, respectively. These trade-offs are becoming less of a barrier to biomedical research as recent advances in optoelectronic microscopy and developments in fluorophore chemistry have enabled scientists to see beyond the diffraction barrier, image deeper into live specimens, and acquire images at unprecedented speed. Selective plane illumination microscopy has provided significant gains in the spatial and temporal acquisition of fluorescence specimens several mm in thickness. With commercial systems now available, this method promises to expand on recent advances in 2-photon deep-tissue imaging with improved speed and reduced photobleaching compared to laser scanning confocal microscopy. Superresolution microscopes are also available in several modalities and can be coupled with selective plane illumination techniques. The combination of methods to increase resolution, acquisition speed, and depth of collection are now being married to common microscope systems, enabling scientists to make significant advances in live cell and in situ imaging in real time. We show that light sheet microscopy provides significant advantages for imaging live zebrafish embryos compared to laser scanning confocal microscopy. PMID:24600334

Applications of fluorescence spectroscopy to problems of food safety: detection of fecal contamination and of the presence of central nervous system tissue and diagnosis of neurological disease

NASA Astrophysics Data System (ADS)

Adhikary, Ramkrishna; Bose, Sayantan; Casey, Thomas A.; Gapsch, Al; Rasmussen, Mark A.; Petrich, Jacob W.

2010-02-01

Applications of fluorescence spectroscopy that enable the real-time or rapid detection of fecal contamination on beef carcasses and the presence of central nervous system tissue in meat products are discussed. The former is achieved by employing spectroscopic signatures of chlorophyll metabolites; the latter, by exploiting the characteristic structure and intensity of lipofuscin in central nervous system tissue. The success of these techniques has led us to investigate the possibility of diagnosing scrapie in sheep by obtaining fluorescence spectra of the retina. Crucial to this diagnosis is the ability to obtain baseline correlations of lipofuscin fluorescence with age. A murine model was employed as a proof of principle of this correlation.

Sample-averaged biexciton quantum yield measured by solution-phase photon correlation.

PubMed

Beyler, Andrew P; Bischof, Thomas S; Cui, Jian; Coropceanu, Igor; Harris, Daniel K; Bawendi, Moungi G

2014-12-10

The brightness of nanoscale optical materials such as semiconductor nanocrystals is currently limited in high excitation flux applications by inefficient multiexciton fluorescence. We have devised a solution-phase photon correlation measurement that can conveniently and reliably measure the average biexciton-to-exciton quantum yield ratio of an entire sample without user selection bias. This technique can be used to investigate the multiexciton recombination dynamics of a broad scope of synthetically underdeveloped materials, including those with low exciton quantum yields and poor fluorescence stability. Here, we have applied this method to measure weak biexciton fluorescence in samples of visible-emitting InP/ZnS and InAs/ZnS core/shell nanocrystals, and to demonstrate that a rapid CdS shell growth procedure can markedly increase the biexciton fluorescence of CdSe nanocrystals.

Vectorized data acquisition and fast triple-correlation integrals for Fluorescence Triple Correlation Spectroscopy

PubMed Central

Ridgeway, William K; Millar, David P; Williamson, James R

2013-01-01

Fluorescence Correlation Spectroscopy (FCS) is widely used to quantitate reaction rates and concentrations of molecules in vitro and in vivo. We recently reported Fluorescence Triple Correlation Spectroscopy (F3CS), which correlates three signals together instead of two. F3CS can analyze the stoichiometries of complex mixtures and detect irreversible processes by identifying time-reversal asymmetries. Here we report the computational developments that were required for the realization of F3CS and present the results as the Triple Correlation Toolbox suite of programs. Triple Correlation Toolbox is a complete data analysis pipeline capable of acquiring, correlating and fitting large data sets. Each segment of the pipeline handles error estimates for accurate error-weighted global fitting. Data acquisition was accelerated with a combination of off-the-shelf counter-timer chips and vectorized operations on 128-bit registers. This allows desktop computers with inexpensive data acquisition cards to acquire hours of multiple-channel data with sub-microsecond time resolution. Off-line correlation integrals were implemented as a two delay time multiple-tau scheme that scales efficiently with multiple processors and provides an unprecedented view of linked dynamics. Global fitting routines are provided to fit FCS and F3CS data to models containing up to ten species. Triple Correlation Toolbox is a complete package that enables F3CS to be performed on existing microscopes. PMID:23525193

Analyzing the performance of fluorescence parameters in the monitoring of leaf nitrogen content of paddy rice

PubMed Central

Yang, Jian; Gong, Wei; Shi, Shuo; Du, Lin; Sun, Jia; Song, Shalei; Chen, Biwu; Zhang, Zhenbing

2016-01-01

Leaf nitrogen content (LNC) is a significant factor which can be utilized to monitor the status of paddy rice and it requires a reliable approach for fast and precise quantification. This investigation aims to quantitatively analyze the correlation between fluorescence parameters and LNC based on laser-induced fluorescence (LIF) technology. The fluorescence parameters exhibited a consistent positive linear correlation with LNC in different growing years (2014 and 2015) and different rice cultivars. The R2 of the models varied from 0.6978 to 0.9045. Support vector machine (SVM) was then utilized to verify the feasibility of the fluorescence parameters for monitoring LNC. Comparison of the fluorescence parameters indicated that F740 is the most sensitive (the R2 of linear regression analysis of the between predicted and measured values changed from 0.8475 to 0.9226, and REs ranged from 3.52% to 4.83%) to the changes in LNC among all fluorescence parameters. Experimental results demonstrated that fluorescence parameters based on LIF technology combined with SVM is a potential method for realizing real-time, non-destructive monitoring of paddy rice LNC, which can provide guidance for the decision-making of farmers in their N fertilization strategies. PMID:27350029

Ethidium bromide as a marker of mtDNA replication in living cells

NASA Astrophysics Data System (ADS)

Villa, Anna Maria; Fusi, Paola; Pastori, Valentina; Amicarelli, Giulia; Pozzi, Chiara; Adlerstein, Daniel; Doglia, Silvia Maria

2012-04-01

Mitochondrial DNA (mtDNA) in tumor cells was found to play an important role in maintaining the malignant phenotype. Using laser scanning confocal fluorescence microscopy (LSCFM) in a recent work, we reported a variable fluorescence intensity of ethidium bromide (EB) in mitochondria nucleoids of living carcinoma cells. Since when EB is bound to nucleic acids its fluorescence is intensified; a higher EB fluorescence intensity could reflect a higher DNA accessibility to EB, suggesting a higher mtDNA replication activity. To prove this hypothesis, in the present work we studied, by LSCFM, the EB fluorescence in mitochondria nucleoids of living neuroblastoma cells, a model system in which differentiation affects the level of mtDNA replication. A drastic decrease of fluorescence was observed after differentiation. To correlate EB fluorescence intensity to the mtDNA replication state, we evaluated the mtDNA nascent strands content by ligation-mediated real-time PCR, and we found a halved amount of replicating mtDNA molecules in differentiating cells. A similar result was obtained by BrdU incorporation. These results indicate that the low EB fluorescence of nucleoids in differentiated cells is correlated to a low content of replicating mtDNA, suggesting that EB may be used as a marker of mtDNA replication in living cells.

CMOS Time-Resolved, Contact, and Multispectral Fluorescence Imaging for DNA Molecular Diagnostics

PubMed Central

Guo, Nan; Cheung, Ka Wai; Wong, Hiu Tung; Ho, Derek

2014-01-01

Instrumental limitations such as bulkiness and high cost prevent the fluorescence technique from becoming ubiquitous for point-of-care deoxyribonucleic acid (DNA) detection and other in-field molecular diagnostics applications. The complimentary metal-oxide-semiconductor (CMOS) technology, as benefited from process scaling, provides several advanced capabilities such as high integration density, high-resolution signal processing, and low power consumption, enabling sensitive, integrated, and low-cost fluorescence analytical platforms. In this paper, CMOS time-resolved, contact, and multispectral imaging are reviewed. Recently reported CMOS fluorescence analysis microsystem prototypes are surveyed to highlight the present state of the art. PMID:25365460

Genetically encoded sensors and fluorescence microscopy for anticancer research

NASA Astrophysics Data System (ADS)

Zagaynova, Elena V.; Shirmanova, Marina V.; Sergeeva, Tatiana F.; Klementieva, Natalia V.; Mishin, Alexander S.; Gavrina, Alena I.; Zlobovskay, Olga A.; Furman, Olga E.; Dudenkova, Varvara V.; Perelman, Gregory S.; Lukina, Maria M.; Lukyanov, Konstantin A.

2017-02-01

Early response of cancer cells to chemical compounds and chemotherapeutic drugs were studied using novel fluorescence tools and microscopy techniques. We applied confocal microscopy, two-photon fluorescence lifetime imaging microscopy and super-resolution localization-based microscopy to assess structural and functional changes in cancer cells in vitro. The dynamics of energy metabolism, intracellular pH, caspase-3 activation during staurosporine-induced apoptosis as well as actin cytoskeleton rearrangements under chemotherapy were evaluated. We have showed that new genetically encoded sensors and advanced fluorescence microscopy methods provide an efficient way for multiparameter analysis of cell activities

X-ray Fluorescence Holography: Principles, Apparatus, and Applications

NASA Astrophysics Data System (ADS)

Hayashi, Kouichi; Korecki, Pawel

2018-06-01

X-ray fluorescence holography (XFH) is an atomic structure determination technique that combines the capabilities of X-ray diffraction and X-ray fluorescence spectroscopy. It provides a unique means of gaining fully three-dimensional information about the local atomic structure and lattice site positions of selected elements inside compound samples. In this work, we discuss experimental and theoretical aspects that are essential for the efficient recording and analysis of X-ray fluorescence holograms and review the most recent advances in XFH. We describe experiments performed with brilliant synchrotron radiation as well as with tabletop setups that employ conventional X-ray tubes.

Ambient measurements of biological aerosol particles near Killarney, Ireland: a comparison between real-time fluorescence and microscopy techniques

NASA Astrophysics Data System (ADS)

Healy, D. A.; Huffman, J. A.; O'Connor, D. J.; Pöhlker, C.; Pöschl, U.; Sodeau, J. R.

2014-08-01

Primary biological aerosol particles (PBAPs) can contribute significantly to the coarse particle burden in many environments. PBAPs can thus influence climate and precipitation systems as cloud nuclei and can spread disease to humans, animals, and plants. Measurement data and techniques for PBAPs in natural environments at high time- and size resolution are, however, sparse, and so large uncertainties remain in the role that biological particles play in the Earth system. In this study two commercial real-time fluorescence particle sensors and a Sporewatch single-stage particle impactor were operated continuously from 2 August to 2 September 2010 at a rural sampling location in Killarney National Park in southwestern Ireland. A cascade impactor was operated periodically to collect size-resolved particles during exemplary periods. Here we report the first ambient comparison of a waveband integrated bioaerosol sensor (WIBS-4) with a ultraviolet aerodynamic particle sizer (UV-APS) and also compare these real-time fluorescence techniques with results of fluorescence and optical microscopy of impacted samples. Both real-time instruments showed qualitatively similar behavior, with increased fluorescent bioparticle concentrations at night, when relative humidity was highest and temperature was lowest. The fluorescent particle number from the FL3 channel of the WIBS-4 and from the UV-APS were strongly correlated and dominated by a 3 μm mode in the particle size distribution. The WIBS FL2 channel exhibited particle modes at approx. 1 and 3 μm, and each was correlated with the concentration of fungal spores commonly observed in air samples collected at the site (ascospores, basidiospores, Ganoderma spp.). The WIBS FL1 channel exhibited variable multimodal distributions turning into a broad featureless single mode after averaging, and exhibited poor correlation with fungal spore concentrations, which may be due to the detection of bacterial and non-biological fluorescent particles. Cladosporium spp., which are among the most abundant fungal spores in many terrestrial environments, were not correlated with any of the real-time fluorescence channels, suggesting that the real-time fluorescence instruments are relatively insensitive to PBAP classes with dark, highly absorptive cell walls. Fluorescence microscopy images of cascade impactor plates showed large numbers of coarse-mode particles consistent with the morphology and weak fluorescence expected of sea salt. Some of these particles were attached to biological cells, suggesting that a marine source influenced the PBAPs observed at the site and that the ocean may be an important contributor to PBAP loadings in coastal environments.

Ambient measurements of biological aerosol particles near Killarney, Ireland: a comparison between real-time fluorescence and microscopy techniques

NASA Astrophysics Data System (ADS)

Healy, D. A.; Huffman, J. A.; O'Connor, D. J.; Pöhlker, C.; Pöschl, U.; Sodeau, J. R.

2014-02-01

Primary biological aerosol particles (PBAP) can contribute significantly to the coarse particle burden in many environments, may thus influence climate and precipitation systems as cloud nuclei, and can spread disease to humans, animals, and plants. Measurements of PBAP in natural environments taken at high time- and size- resolution are, however, sparse and so large uncertainties remain in the role that biological particles play in the Earth system. In this study two commercial real-time fluorescence particle sensors and a Sporewatch single-stage particle impactor were operated continuously from 2 August to 2 September 2010 at a rural sampling location in Killarney National Park in south western Ireland. A cascade impactor was operated periodically to collect size-resolved particles during exemplary periods. Here we report the first ambient comparison of the waveband integrated bioaerosol sensor (WIBS-4) with the ultraviolet aerodynamic particle sizer (UV-APS) and also compare these real-time fluorescence techniques with results of fluorescence and optical microscopy of impacted samples. Both real-time instruments showed qualitatively similar behaviour, with increased fluorescent bioparticle concentrations at night when relative humidity was highest and temperature was lowest. The fluorescent particle number from the FL3 channel of the WIBS-4 and from the UV-APS were strongly correlated and dominated by a 3 μm mode in the particle size distribution. The WIBS FL2 channel exhibited particle modes at approx. 1 and 3 μm, and each were correlated with the concentration of fungal spores commonly observed in air samples collected at the site (ascospores, basidiospores, Ganoderma spp.). The WIBS FL1 channel exhibited variable multi-modal distributions turning into a broad featureless single mode after averaging and exhibited poor correlation with fungal spore concentrations, which may be due to the detection of bacterial and non-biological fluorescent particles. Cladosporium spp., which are among the most abundant fungal spores in many terrestrial environments, were not correlated with any of the real-time fluorescence channels, suggesting that the real-time fluorescence instruments are insensitive to PBAP classes with dark, highly absorptive cell walls. Fluorescence microscopy images of cascade impactor plates showed large numbers of coarse mode particles consistent with the morphology and weak fluorescence expected of sea salt. Some of these particles were attached to biological cells, suggesting that a marine source influenced the PBAP observed at the site and that the ocean may be an important contributor to PBAP loadings in coastal environments.

Diffusion behavior of the fluorescent proteins eGFP and Dreiklang in solvents of different viscosity monitored by fluorescence correlation spectroscopy

NASA Astrophysics Data System (ADS)

Junghans, Cornelia; Schmitt, Franz-Josef; Vukojević, Vladana; Friedrich, Thomas

2016-12-01

Fluorescence correlation spectroscopy relies on temporal autocorrelation analysis of fluorescence intensity fluctuations that spontaneously arise in systems at equilibrium due to molecular motion and changes of state that cause changes in fluorescence, such as triplet state transition, photoisomerization and other photophysical transformations, to determine the rates of these processes. The stability of a fluorescent molecule against dark state conversion is of particular concern for chromophores intended to be used as reference tags for comparing diffusion processes on multiple time scales. In this work, we analyzed properties of two fluorescent proteins, the photoswitchable Dreiklang and its parental eGFP, in solvents of different viscosity to vary the diffusion time through the observation volume element by several orders of magnitude. In contrast to eGFP, Dreiklang undergoes a dark-state conversion on the time scale of tens to hundreds of microseconds under conditions of intense fluorescence excitation, which results in artificially shortened diffusion times if the diffusional motion through the observation volume is sufficiently slowed down. Such photophysical quenching processes have also been observed in FCS studies on other photoswitchable fluorescent proteins including Citrine, from which Dreiklang was derived by genetic engineering. This property readily explains the discrepancies observed previously between the diffusion times of eGFP- and Dreiklang-labeled plasma membrane protein complexes.

[Retrospective, descriptive, observational study of treatment of multiple actinic keratoses with topical methyl aminolevulinate and red light: results in clinical practice and correlation with fluorescence imaging].

PubMed

Fernández-Guarino, M; Harto, A; Sánchez-Ronco, M; Pérez-García, B; Marquet, A; Jaén, P

2008-12-01

Actinic keratosis (AK) is one of the most common skin diseases seen in clinical practice. In the last 5 years, several studies assessing the efficacy of photodynamic therapy in the treatment of multiple AKs have been published. We aimed to assess the clinical outcomes of photodynamic therapy in patients with multiple AKs and the correlation of those outcomes with fluorescence imaging. In this retrospective, descriptive, observational study of 57 patients treated in our hospital with photodynamic therapy for multiple AKs, we recorded age, sex, and lesion site (face, scalp, and dorsum of the hands). All patients were treated in the same way: methyl aminolevulinic acid (Metvix) was applied for 3 hours and the skin then irradiated with red light at 630 nm, 37 J/cm(2), for 7.5 minutes (Aktilite). The response, remission duration, tolerance, number of sessions, and fluorescence images were recorded by site. The chi(2) test was used to assess between-site differences and the correlation between fluorescence imaging and clinical response. The greatest improvements were obtained for facial lesions; these required fewer sessions and remission lasted longer than lesions at other sites. The treatment was best tolerated on the dorsum of the hands. The fluorescence area and the reduction in intensity on applying treatment were found to be strongly and significantly correlated with the extent of clinical response. Overall, the outcomes of treatment of multiple AKs with photodynamic therapy are better for the face than for the scalp and dorsum of the hands. Fluorescence imaging may be an effective tool for predicting response to treatment.

Correlation between Wavelength Dispersive X-ray Fluorescence (WDXRF) analysis of hardened concrete for chlorides vs. Atomic Absorption (AA) analysis in accordance with AASHTO T- 260; sampling and testing for chloride ion in concrete and concrete raw mater

DOT National Transportation Integrated Search

2014-04-01

A correlation between Wavelength Dispersive X-ray Fluorescence(WDXRF) analysis of Hardened : Concrete for Chlorides and Atomic Absorption (AA) analysis (current method AASHTO T-260, procedure B) has been : found and a new method of analysis has been ...

Processes Affecting Variability of Fluorescence Signals from Benthic Targets in Shallow Waters

DTIC Science & Technology

1997-09-30

processes in the Department of Chemistry at Brookhaven National Laboratory. The model organisms used are primarily cultured zooxanthellae obtained from...and closed (Fm) photosystem II reaction centers in the zooxanthellae isolated from the fire coral, Montipora. The short lifetime curve corresponds...individual zooxanthellae strains, is highly correlated Figure 2. The correlation between the average fluorescence lifetimes, calculated from a four

Correlative cryo-fluorescence light microscopy and cryo-electron tomography of Streptomyces.

PubMed

Koning, Roman I; Celler, Katherine; Willemse, Joost; Bos, Erik; van Wezel, Gilles P; Koster, Abraham J

2014-01-01

Light microscopy and electron microscopy are complementary techniques that in a correlative approach enable identification and targeting of fluorescently labeled structures in situ for three-dimensional imaging at nanometer resolution. Correlative imaging allows electron microscopic images to be positioned in a broader temporal and spatial context. We employed cryo-correlative light and electron microscopy (cryo-CLEM), combining cryo-fluorescence light microscopy and cryo-electron tomography, on vitrified Streptomyces bacteria to study cell division. Streptomycetes are mycelial bacteria that grow as long hyphae and reproduce via sporulation. On solid media, Streptomyces subsequently form distinct aerial mycelia where cell division leads to the formation of unigenomic spores which separate and disperse to form new colonies. In liquid media, only vegetative hyphae are present divided by noncell separating crosswalls. Their multicellular life style makes them exciting model systems for the study of bacterial development and cell division. Complex intracellular structures have been visualized with transmission electron microscopy. Here, we describe the methods for cryo-CLEM that we applied for studying Streptomyces. These methods include cell growth, fluorescent labeling, cryo-fixation by vitrification, cryo-light microscopy using a Linkam cryo-stage, image overlay and relocation, cryo-electron tomography using a Titan Krios, and tomographic reconstruction. Additionally, methods for segmentation, volume rendering, and visualization of the correlative data are described. © 2014 Elsevier Inc. All rights reserved.

Pentosidine formation in skin correlates with severity of complications in individuals with long-standing IDDM.

PubMed

Sell, D R; Lapolla, A; Odetti, P; Fogarty, J; Monnier, V M

1992-10-01

Pentosidine is an advanced glycosylation end product and protein cross-link that results from the reaction of pentoses with proteins. Recent data indicate that long-term glycation of proteins with glucose also leads to pentosidine formation through sugar fragmentation. In this study, the relationship between the severity of diabetic complications and pentosidine formation was investigated in collagen from skin-punch biopsies from 25 nondiabetic control subjects and 41 IDDM patients with diabetes duration greater than 17 yr. Pentosidine was significantly elevated in all IDDM patients versus control subjects (P less than 0.0001). It correlated strongly with age (P less than 0.0001) and weakly with duration (P less than 0.082). Age-adjusted pentosidine levels were highest in grade 2 (severe) versus grade 1 and 0 complication in all four parameters tested (retinopathy, proteinuria, arterial stiffness, and joint stiffness). Significant differences were found for retinopathy (P less than 0.014) and joint stiffness (P less than 0.041). The highest degree of association was with the cumulative grade of individual complication (P less than 0.005), determined by summing indexes of all four parameters. Pentosidine also was significantly elevated in the serum of IDDM patients compared with control subjects (P less than 0.0001), but levels were not significantly correlated with age, diabetes duration, complication, or skin collagen pentosidine (P greater than 0.05). A high correlation between pentosidine levels and long-wave collagen-linked fluorescence also was observed, suggesting that pentosidine is a generalized marker of accelerated tissue modification by the advanced glycosylation/Maillard reaction, which is enhanced in IDDM patients with severe complications.

Correlation and classification of single kernel fluorescence hyperspectral data with aflatoxin concentration in corn kernels inoculated with Aspergillus flavus spores.

PubMed

Yao, H; Hruska, Z; Kincaid, R; Brown, R; Cleveland, T; Bhatnagar, D

2010-05-01

The objective of this study was to examine the relationship between fluorescence emissions of corn kernels inoculated with Aspergillus flavus and aflatoxin contamination levels within the kernels. Aflatoxin contamination in corn has been a long-standing problem plaguing the grain industry with potentially devastating consequences to corn growers. In this study, aflatoxin-contaminated corn kernels were produced through artificial inoculation of corn ears in the field with toxigenic A. flavus spores. The kernel fluorescence emission data were taken with a fluorescence hyperspectral imaging system when corn kernels were excited with ultraviolet light. Raw fluorescence image data were preprocessed and regions of interest in each image were created for all kernels. The regions of interest were used to extract spectral signatures and statistical information. The aflatoxin contamination level of single corn kernels was then chemically measured using affinity column chromatography. A fluorescence peak shift phenomenon was noted among different groups of kernels with different aflatoxin contamination levels. The fluorescence peak shift was found to move more toward the longer wavelength in the blue region for the highly contaminated kernels and toward the shorter wavelengths for the clean kernels. Highly contaminated kernels were also found to have a lower fluorescence peak magnitude compared with the less contaminated kernels. It was also noted that a general negative correlation exists between measured aflatoxin and the fluorescence image bands in the blue and green regions. The correlation coefficients of determination, r(2), was 0.72 for the multiple linear regression model. The multivariate analysis of variance found that the fluorescence means of four aflatoxin groups, <1, 1-20, 20-100, and >or=100 ng g(-1) (parts per billion), were significantly different from each other at the 0.01 level of alpha. Classification accuracy under a two-class schema ranged from 0.84 to 0.91 when a threshold of either 20 or 100 ng g(-1) was used. Overall, the results indicate that fluorescence hyperspectral imaging may be applicable in estimating aflatoxin content in individual corn kernels.

Understanding Zinc Quantification with Existing and Advanced Ditopic Fluorescent Zinpyr Sensors

PubMed Central

Buccella, Daniela; Horowitz, Joshua A.; Lippard, Stephen J.

2011-01-01

Treatment of aqueous zinc solutions with incremental additions of a ditopic fluorescent sensor of the Zinpyr family, based on pyridine/pyrazine-containing metal recognition units, affords a fluorescence titration curve with a sharp maximum at a sensor:Zn2+ ratio of 0.5 (Zhang, X-a.; Hayes, D.; Smith, S. J.; Friedle, S.; Lippard, S. J. J. Am. Chem Soc. 2008, 130, 15788–15789). This fluorescence response enables the quantification of readily chelatable zinc in biological samples by a simple titration protocol. In the present work a new set of ditopic fluorescence zinc sensors functionalized with pyridine/pyrazine-containing metal chelating units is described, and through detailed studies the principles governing the characteristic “OFF-ON-OFF” fluorescence behavior and quantification capabilities of the family are delineated. Incorporation of carboxylate/ester groups in the 6 position of the fluorescein allows for control of the spatial distribution of the sensor for selective extra- or intracellular imaging of mobile zinc, without introducing significant changes in zinc-binding properties. A combination of spectrophotometric and potentiometric measurements provided a complete description of the H+ and Zn2+ binding properties of the compounds and their correlation with the observed fluorescence profile. The first zinc-binding event has an apparent affinity, K1′, of 1.9–3.1×109 M−1, whereas for coordination of the second Zn2+ ion, responsible for fluorescence turn on, the apparent formation constant K2′ is 5.5–6.9×107 M−1. A detailed chemical and mathematical analysis of the system demonstrated that the difference in emission efficiencies of the dimetalated (LZn2) vs. monometalated (LZn) and metal free (L) forms, a consequence of the combined quenching effects of the two metal-chelating units, significantly influences the shape of the titration curve. The scope of the titration method was investigated mathematically, and a lower boundary for the range of concentrations that can be determined was established as a function of the magnitude of K2′. Our results suggest that the principles governing the response of the ZPP1 series are applicable to other analogues of the Zinpyr family. Moreover, they may guide the design of other ditopic sensors suitable for determining the concentrations of a wide range of mobile metal ions and other chemical signaling agents of relevance in biological systems. PMID:21351756

Estimating the Biodegradability of Treated Sewage Samples Using Synchronous Fluorescence Spectra

PubMed Central

Lai, Tien M.; Shin, Jae-Ki; Hur, Jin

2011-01-01

Synchronous fluorescence spectra (SFS) and the first derivative spectra of the influent versus the effluent wastewater samples were compared and the use of fluorescence indices is suggested as a means to estimate the biodegradability of the effluent wastewater. Three distinct peaks were identified from the SFS of the effluent wastewater samples. Protein-like fluorescence (PLF) was reduced, whereas fulvic and/or humic-like fluorescence (HLF) were enhanced, suggesting that the two fluorescence characteristics may represent biodegradable and refractory components, respectively. Five fluorescence indices were selected for the biodegradability estimation based on the spectral features changing from the influent to the effluent. Among the selected indices, the relative distribution of PLF to the total fluorescence area of SFS (Index II) exhibited the highest correlation coefficient with total organic carbon (TOC)-based biodegradability, which was even higher than those obtained with the traditional oxygen demand-based parameters. A multiple regression analysis using Index II and the area ratio of PLF to HLF (Index III) demonstrated the enhancement of the correlations from 0.558 to 0.711 for TOC-based biodegradability. The multiple regression equation finally obtained was 0.148 × Index II − 4.964 × Index III − 0.001 and 0.046 × Index II − 1.128 × Index III + 0.026. The fluorescence indices proposed here are expected to be utilized for successful development of real-time monitoring using a simple fluorescence sensing device for the biodegradability of treated sewage. PMID:22164023

Estimating the biodegradability of treated sewage samples using synchronous fluorescence spectra.

PubMed

Lai, Tien M; Shin, Jae-Ki; Hur, Jin

2011-01-01

Synchronous fluorescence spectra (SFS) and the first derivative spectra of the influent versus the effluent wastewater samples were compared and the use of fluorescence indices is suggested as a means to estimate the biodegradability of the effluent wastewater. Three distinct peaks were identified from the SFS of the effluent wastewater samples. Protein-like fluorescence (PLF) was reduced, whereas fulvic and/or humic-like fluorescence (HLF) were enhanced, suggesting that the two fluorescence characteristics may represent biodegradable and refractory components, respectively. Five fluorescence indices were selected for the biodegradability estimation based on the spectral features changing from the influent to the effluent. Among the selected indices, the relative distribution of PLF to the total fluorescence area of SFS (Index II) exhibited the highest correlation coefficient with total organic carbon (TOC)-based biodegradability, which was even higher than those obtained with the traditional oxygen demand-based parameters. A multiple regression analysis using Index II and the area ratio of PLF to HLF (Index III) demonstrated the enhancement of the correlations from 0.558 to 0.711 for TOC-based biodegradability. The multiple regression equation finally obtained was 0.148 × Index II - 4.964 × Index III - 0.001 and 0.046 × Index II - 1.128 × Index III + 0.026. The fluorescence indices proposed here are expected to be utilized for successful development of real-time monitoring using a simple fluorescence sensing device for the biodegradability of treated sewage.

Mapping Diffusion in a Living Cell via the Phasor Approach

PubMed Central

Ranjit, Suman; Lanzano, Luca; Gratton, Enrico

2014-01-01

Diffusion of a fluorescent protein within a cell has been measured using either fluctuation-based techniques (fluorescence correlation spectroscopy (FCS) or raster-scan image correlation spectroscopy) or particle tracking. However, none of these methods enables us to measure the diffusion of the fluorescent particle at each pixel of the image. Measurement using conventional single-point FCS at every individual pixel results in continuous long exposure of the cell to the laser and eventual bleaching of the sample. To overcome this limitation, we have developed what we believe to be a new method of scanning with simultaneous construction of a fluorescent image of the cell. In this believed new method of modified raster scanning, as it acquires the image, the laser scans each individual line multiple times before moving to the next line. This continues until the entire area is scanned. This is different from the original raster-scan image correlation spectroscopy approach, where data are acquired by scanning each frame once and then scanning the image multiple times. The total time of data acquisition needed for this method is much shorter than the time required for traditional FCS analysis at each pixel. However, at a single pixel, the acquired intensity time sequence is short; requiring nonconventional analysis of the correlation function to extract information about the diffusion. These correlation data have been analyzed using the phasor approach, a fit-free method that was originally developed for analysis of FLIM images. Analysis using this method results in an estimation of the average diffusion coefficient of the fluorescent species at each pixel of an image, and thus, a detailed diffusion map of the cell can be created. PMID:25517145

Fluorescent kapakahines serve as non-toxic probes for live cell Golgi imaging.

PubMed

Rocha, Danilo D; Espejo, Vinson R; Rainier, Jon D; La Clair, James J; Costa-Lotufo, Letícia V

2015-09-01

There is an ongoing need for fluorescent probes that specifically-target select organelles within mammalian cells. This study describes the development of probes for the selective labeling of the Golgi apparatus and offers applications for live cell and fixed cell imaging. The kapakahines, characterized by a common C(3)-N(1') dimeric tryptophan linkage, comprise a unique family of bioactive marine depsipeptide natural products. We describe the uptake and subcellular localization of fluorescently-labeled analogs of kapakahine E. Using confocal microscopy, we identify a rapid and selective localization within the Golgi apparatus. Comparison with commercial Golgi stains indicates a unique localization pattern, which differs from currently available materials, therein offering a new tool to monitor the Golgi in live cells without toxic side effects. This study identifies a fluorescent analog of kapakahine E that is rapidly uptaken in cells and localizes within the Golgi apparatus. The advance of microscopic methods is reliant on the parallel discovery of next generation molecular probes. This study describes the advance of stable and viable probe for staining the Golgi apparatus. Copyright © 2015 Elsevier Inc. All rights reserved.

Beyond the margins: real-time detection of cancer using targeted fluorophores

PubMed Central

Zhang, Ray R.; Schroeder, Alexandra B.; Grudzinski, Joseph J.; Rosenthal, Eben L.; Warram, Jason M.; Pinchuk, Anatoly N.; Eliceiri, Kevin W.; Kuo, John S.; Weichert, Jamey P.

2017-01-01

Over the past two decades, synergistic innovations in imaging technology have resulted in a revolution in which a range of biomedical applications are now benefiting from fluorescence imaging. Specifically, advances in fluorophore chemistry and imaging hardware, and the identification of targetable biomarkers have now positioned intraoperative fluorescence as a highly specific real-time detection modality for surgeons in oncology. In particular, the deeper tissue penetration and limited autofluorescence of near-infrared (NIR) fluorescence imaging improves the translational potential of this modality over visible-light fluorescence imaging. Rapid developments in fluorophores with improved characteristics, detection instrumentation, and targeting strategies led to the clinical testing in the early 2010s of the first targeted NIR fluorophores for intraoperative cancer detection. The foundations for the advances that underline this technology continue to be nurtured by the multidisciplinary collaboration of chemists, biologists, engineers, and clinicians. In this Review, we highlight the latest developments in NIR fluorophores, cancer-targeting strategies, and detection instrumentation for intraoperative cancer detection, and consider the unique challenges associated with their effective application in clinical settings. PMID:28094261

Characterizing harmful advanced glycation end-products (AGEs) and ribosylated aggregates of yellow mustard seed phytocystatin: Effects of different monosaccharides.

PubMed

Ahmed, Azaj; Shamsi, Anas; Bano, Bilqees

2017-01-15

Advanced glycation end products (AGEs) are at the core of variety of diseases ranging from diabetes to renal failure and hence gaining wide consideration. This study was aimed at characterizing the AGEs of phytocystatin isolated from mustard seeds (YMP) when incubated with different monosaccharides (glucose, ribose and mannose) using fluorescence, ultraviolet, circular dichroism (CD) spectroscopy and microscopy. Ribose was found to be the most potent glycating agent as evident by AGEs specific fluorescence and absorbance. YMP exists as a molten globule like structure on day 24 as depicted by high ANS fluorescence and altered intrinsic fluorescence. Glycated YMP as AGEs and ribose induced aggregates were observed at day 28 and 32 respectively. In our study we have also examined the anti-aggregative potential of polyphenol, resveratrol. Our results suggested the anti-aggregative behavior of resveratrol as it prevented the in vitro aggregation of YMP, although further studies are required to decode the mechanism by which resveratrol prevents the aggregation. Copyright © 2016 Elsevier B.V. All rights reserved.

Determination of Dynamics of Plant Plasma Membrane Proteins with Fluorescence Recovery and Raster Image Correlation Spectroscopy.

PubMed

Laňková, Martina; Humpolíčková, Jana; Vosolsobě, Stanislav; Cit, Zdeněk; Lacek, Jozef; Čovan, Martin; Čovanová, Milada; Hof, Martin; Petrášek, Jan

2016-04-01

A number of fluorescence microscopy techniques are described to study dynamics of fluorescently labeled proteins, lipids, nucleic acids, and whole organelles. However, for studies of plant plasma membrane (PM) proteins, the number of these techniques is still limited because of the high complexity of processes that determine the dynamics of PM proteins and the existence of cell wall. Here, we report on the usage of raster image correlation spectroscopy (RICS) for studies of integral PM proteins in suspension-cultured tobacco cells and show its potential in comparison with the more widely used fluorescence recovery after photobleaching method. For RICS, a set of microscopy images is obtained by single-photon confocal laser scanning microscopy (CLSM). Fluorescence fluctuations are subsequently correlated between individual pixels and the information on protein mobility are extracted using a model that considers processes generating the fluctuations such as diffusion and chemical binding reactions. As we show here using an example of two integral PM transporters of the plant hormone auxin, RICS uncovered their distinct short-distance lateral mobility within the PM that is dependent on cytoskeleton and sterol composition of the PM. RICS, which is routinely accessible on modern CLSM instruments, thus represents a valuable approach for studies of dynamics of PM proteins in plants.

Exploring the Dynamics of Cell Processes through Simulations of Fluorescence Microscopy Experiments

PubMed Central

Angiolini, Juan; Plachta, Nicolas; Mocskos, Esteban; Levi, Valeria

2015-01-01

Fluorescence correlation spectroscopy (FCS) methods are powerful tools for unveiling the dynamical organization of cells. For simple cases, such as molecules passively moving in a homogeneous media, FCS analysis yields analytical functions that can be fitted to the experimental data to recover the phenomenological rate parameters. Unfortunately, many dynamical processes in cells do not follow these simple models, and in many instances it is not possible to obtain an analytical function through a theoretical analysis of a more complex model. In such cases, experimental analysis can be combined with Monte Carlo simulations to aid in interpretation of the data. In response to this need, we developed a method called FERNET (Fluorescence Emission Recipes and Numerical routines Toolkit) based on Monte Carlo simulations and the MCell-Blender platform, which was designed to treat the reaction-diffusion problem under realistic scenarios. This method enables us to set complex geometries of the simulation space, distribute molecules among different compartments, and define interspecies reactions with selected kinetic constants, diffusion coefficients, and species brightness. We apply this method to simulate single- and multiple-point FCS, photon-counting histogram analysis, raster image correlation spectroscopy, and two-color fluorescence cross-correlation spectroscopy. We believe that this new program could be very useful for predicting and understanding the output of fluorescence microscopy experiments. PMID:26039162

Correlative Electron and Fluorescence Microscopy of Magnetotactic Bacteria in Liquid: Toward In Vivo Imaging

PubMed Central

Woehl, Taylor J.; Kashyap, Sanjay; Firlar, Emre; Perez-Gonzalez, Teresa; Faivre, Damien; Trubitsyn, Denis; Bazylinski, Dennis A.; Prozorov, Tanya

2014-01-01

Magnetotactic bacteria biomineralize ordered chains of uniform, membrane-bound magnetite or greigite nanocrystals that exhibit nearly perfect crystal structures and species-specific morphologies. Transmission electron microscopy (TEM) is a critical technique for providing information regarding the organization of cellular and magnetite structures in these microorganisms. However, conventional TEM can only be used to image air-dried or vitrified bacteria removed from their natural environment. Here we present a correlative scanning TEM (STEM) and fluorescence microscopy technique for imaging viable cells of Magnetospirillum magneticum strain AMB-1 in liquid using an in situ fluid cell TEM holder. Fluorescently labeled cells were immobilized on microchip window surfaces and visualized in a fluid cell with STEM, followed by correlative fluorescence imaging to verify their membrane integrity. Notably, the post-STEM fluorescence imaging indicated that the bacterial cell wall membrane did not sustain radiation damage during STEM imaging at low electron dose conditions. We investigated the effects of radiation damage and sample preparation on the bacteria viability and found that approximately 50% of the bacterial membranes remained intact after an hour in the fluid cell, decreasing to ~30% after two hours. These results represent a first step toward in vivo studies of magnetite biomineralization in magnetotactic bacteria. PMID:25358460

Correlative Electron and Fluorescence Microscopy of Magnetotactic Bacteria in Liquid: Toward In Vivo Imaging

DOE PAGES

Woehl, Taylor J.; Kashyap, Sanjay; Firlar, Emre; ...

2014-10-31

Magnetotactic bacteria biomineralize ordered chains of uniform, membrane-bound magnetite or greigite nanocrystals that exhibit nearly perfect crystal structures and species-specific morphologies. Transmission electron microscopy (TEM) is a critical technique for providing information regarding the organization of cellular and magnetite structures in these microorganisms. However, conventional TEM can only be used to image air-dried or vitrified bacteria removed from their natural environment. Here we present a correlative scanning TEM (STEM) and fluorescence microscopy technique for imaging viable cells of Magnetospirillum magneticum strain AMB-1 in liquid using an in situ fluid cell TEM holder. Fluorescently labeled cells were immobilized on microchip windowmore » surfaces and visualized in a fluid cell with STEM, followed by correlative fluorescence imaging to verify their membrane integrity. Notably, the post-STEM fluorescence imaging indicated that the bacterial cell wall membrane did not sustain radiation damage during STEM imaging at low electron dose conditions. We investigated the effects of radiation damage and sample preparation on the bacteria viability and found that approximately 50% of the bacterial membranes remained intact after an hour in the fluid cell, decreasing to ~30% after two hours. These results represent a first step toward in vivo studies of magnetite biomineralization in magnetotactic bacteria.« less

Detection of proximal caries using quantitative light-induced fluorescence-digital and laser fluorescence: a comparative study.

PubMed

Yoon, Hyung-In; Yoo, Min-Jeong; Park, Eun-Jin

2017-12-01

The purpose of this study was to evaluate the in vitro validity of quantitative light-induced fluorescence-digital (QLF-D) and laser fluorescence (DIAGNOdent) for assessing proximal caries in extracted premolars, using digital radiography as reference method. A total of 102 extracted premolars with similar lengths and shapes were used. A single operator conducted all the examinations using three different detection methods (bitewing radiography, QLF-D, and DIAGNOdent). The bitewing x-ray scale, QLF-D fluorescence loss (ΔF), and DIAGNOdent peak readings were compared and statistically analyzed. Each method showed an excellent reliability. The correlation coefficient between bitewing radiography and QLF-D, DIAGNOdent were -0.644 and 0.448, respectively, while the value between QLF-D and DIAGNOdent was -0.382. The kappa statistics for bitewing radiography and QLF-D had a higher diagnosis consensus than those for bitewing radiography and DIAGNOdent. The QLF-D was moderately to highly accurate (AUC = 0.753 - 0.908), while DIAGNOdent was moderately to less accurate (AUC = 0.622 - 0.784). All detection methods showed statistically significant correlation and high correlation between the bitewing radiography and QLF-D. QLF-D was found to be a valid and reliable alternative diagnostic method to digital bitewing radiography for in vitro detection of proximal caries.

Spectral and Temporal Laser Fluorescence Analysis Such as for Natural Aquatic Environments

NASA Technical Reports Server (NTRS)

Chekalyuk, Alexander (Inventor)

2015-01-01

An Advanced Laser Fluorometer (ALF) can combine spectrally and temporally resolved measurements of laser-stimulated emission (LSE) for characterization of dissolved and particulate matter, including fluorescence constituents, in liquids. Spectral deconvolution (SDC) analysis of LSE spectral measurements can accurately retrieve information about individual fluorescent bands, such as can be attributed to chlorophyll-a (Chl-a), phycobiliprotein (PBP) pigments, or chromophoric dissolved organic matter (CDOM), among others. Improved physiological assessments of photosynthesizing organisms can use SDC analysis and temporal LSE measurements to assess variable fluorescence corrected for SDC-retrieved background fluorescence. Fluorescence assessments of Chl-a concentration based on LSE spectral measurements can be improved using photo-physiological information from temporal measurements. Quantitative assessments of PBP pigments, CDOM, and other fluorescent constituents, as well as basic structural characterizations of photosynthesizing populations, can be performed using SDC analysis of LSE spectral measurements.

Fluorescence-based methods for detecting caries lesions: systematic review, meta-analysis and sources of heterogeneity.

PubMed

Gimenez, Thais; Braga, Mariana Minatel; Raggio, Daniela Procida; Deery, Chris; Ricketts, David N; Mendes, Fausto Medeiros

2013-01-01

Fluorescence-based methods have been proposed to aid caries lesion detection. Summarizing and analysing findings of studies about fluorescence-based methods could clarify their real benefits. We aimed to perform a comprehensive systematic review and meta-analysis to evaluate the accuracy of fluorescence-based methods in detecting caries lesions. Two independent reviewers searched PubMed, Embase and Scopus through June 2012 to identify papers/articles published. Other sources were checked to identify non-published literature. STUDY ELIGIBILITY CRITERIA, PARTICIPANTS AND DIAGNOSTIC METHODS: The eligibility criteria were studies that: (1) have assessed the accuracy of fluorescence-based methods of detecting caries lesions on occlusal, approximal or smooth surfaces, in both primary or permanent human teeth, in the laboratory or clinical setting; (2) have used a reference standard; and (3) have reported sufficient data relating to the sample size and the accuracy of methods. A diagnostic 2×2 table was extracted from included studies to calculate the pooled sensitivity, specificity and overall accuracy parameters (Diagnostic Odds Ratio and Summary Receiver-Operating curve). The analyses were performed separately for each method and different characteristics of the studies. The quality of the studies and heterogeneity were also evaluated. Seventy five studies met the inclusion criteria from the 434 articles initially identified. The search of the grey or non-published literature did not identify any further studies. In general, the analysis demonstrated that the fluorescence-based method tend to have similar accuracy for all types of teeth, dental surfaces or settings. There was a trend of better performance of fluorescence methods in detecting more advanced caries lesions. We also observed moderate to high heterogeneity and evidenced publication bias. Fluorescence-based devices have similar overall performance; however, better accuracy in detecting more advanced caries lesions has been observed.

Challenges for Super-Resolution Localization Microscopy and Biomolecular Fluorescent Nano-Probing in Cancer Research

PubMed Central

Ilić, Nataša; Pilarczyk, Götz; Lee, Jin-Ho; Logeswaran, Abiramy; Borroni, Aurora Paola; Krufczik, Matthias; Theda, Franziska; Waltrich, Nadine; Bestvater, Felix; Hildenbrand, Georg; Cremer, Christoph; Blank, Michael

2017-01-01

Understanding molecular interactions and regulatory mechanisms in tumor initiation, progression, and treatment response are key requirements towards advanced cancer diagnosis and novel treatment procedures in personalized medicine. Beyond decoding the gene expression, malfunctioning and cancer-related epigenetic pathways, investigations of the spatial receptor arrangements in membranes and genome organization in cell nuclei, on the nano-scale, contribute to elucidating complex molecular mechanisms in cells and tissues. By these means, the correlation between cell function and spatial organization of molecules or molecular complexes can be studied, with respect to carcinogenesis, tumor sensitivity or tumor resistance to anticancer therapies, like radiation or antibody treatment. Here, we present several new applications for bio-molecular nano-probes and super-resolution, laser fluorescence localization microscopy and their potential in life sciences, especially in biomedical and cancer research. By means of a tool-box of fluorescent antibodies, green fluorescent protein (GFP) tagging, or specific oligonucleotides, we present tumor relevant re-arrangements of Erb-receptors in membranes, spatial organization of Smad specific ubiquitin protein ligase 2 (Smurf2) in the cytosol, tumor cell characteristic heterochromatin organization, and molecular re-arrangements induced by radiation or antibody treatment. The main purpose of this article is to demonstrate how nano-scaled distance measurements between bio-molecules, tagged by appropriate nano-probes, can be applied to elucidate structures and conformations of molecular complexes which are characteristic of tumorigenesis and treatment responses. These applications open new avenues towards a better interpretation of the spatial organization and treatment responses of functionally relevant molecules, at the single cell level, in normal and cancer cells, offering new potentials for individualized medicine. PMID:28956810

Challenges for Super-Resolution Localization Microscopy and Biomolecular Fluorescent Nano-Probing in Cancer Research.

PubMed

Hausmann, Michael; Ilić, Nataša; Pilarczyk, Götz; Lee, Jin-Ho; Logeswaran, Abiramy; Borroni, Aurora Paola; Krufczik, Matthias; Theda, Franziska; Waltrich, Nadine; Bestvater, Felix; Hildenbrand, Georg; Cremer, Christoph; Blank, Michael

2017-09-28

Understanding molecular interactions and regulatory mechanisms in tumor initiation, progression, and treatment response are key requirements towards advanced cancer diagnosis and novel treatment procedures in personalized medicine. Beyond decoding the gene expression, malfunctioning and cancer-related epigenetic pathways, investigations of the spatial receptor arrangements in membranes and genome organization in cell nuclei, on the nano-scale, contribute to elucidating complex molecular mechanisms in cells and tissues. By these means, the correlation between cell function and spatial organization of molecules or molecular complexes can be studied, with respect to carcinogenesis, tumor sensitivity or tumor resistance to anticancer therapies, like radiation or antibody treatment. Here, we present several new applications for bio-molecular nano-probes and super-resolution, laser fluorescence localization microscopy and their potential in life sciences, especially in biomedical and cancer research. By means of a tool-box of fluorescent antibodies, green fluorescent protein (GFP) tagging, or specific oligonucleotides, we present tumor relevant re-arrangements of Erb-receptors in membranes, spatial organization of Smad specific ubiquitin protein ligase 2 (Smurf2) in the cytosol, tumor cell characteristic heterochromatin organization, and molecular re-arrangements induced by radiation or antibody treatment. The main purpose of this article is to demonstrate how nano-scaled distance measurements between bio-molecules, tagged by appropriate nano-probes, can be applied to elucidate structures and conformations of molecular complexes which are characteristic of tumorigenesis and treatment responses. These applications open new avenues towards a better interpretation of the spatial organization and treatment responses of functionally relevant molecules, at the single cell level, in normal and cancer cells, offering new potentials for individualized medicine.

In situ fluorescence spectroscopy correlates ionomer degradation to reactive oxygen species generation in an operating fuel cell.

PubMed

Prabhakaran, Venkateshkumar; Arges, Christopher G; Ramani, Vijay

2013-11-21

The rate of generation of reactive oxygen species (ROS) within the polymer electrolyte membrane (PEM) of an operating proton exchange member fuel cell (PEMFC) was monitored using in situ fluorescence spectroscopy. A modified barrier layer was introduced between the PEM and the electrocatalyst layer to eliminate metal-dye interactions and fluorescence resonance energy transfer (FRET) effects during measurements. Standard fuel cell operating parameters (temperature, relative humidity, and electrode potential) were systematically varied to evaluate their influence on the rate of ROS generation during PEMFC operation. Independently, the macroscopic rate of PEM degradation was measured by monitoring the fluoride ion emission rate (FER) in the effluent stream at each operating condition. The ROS generation reaction rate constant (estimated from the in situ fluorescence experiments) correlated perfectly with the measured FER across all conditions, demonstrating unequivocally for the first time that a direct correlation exists between in situ ROS generation and PEM macroscopic degradation. The activation energy for ROS generation within the PEM was estimated to be 12.5 kJ mol(-1).

Analysis of laser-induced-fluorescence carbon monoxide measurements in turbulent nonpremixed flames

NASA Astrophysics Data System (ADS)

Mokhov, A. V.; Levinsky, H. B.; van der Meij, C. E.; Jacobs, R. A. A. M.

1995-10-01

The influence of fluctuating concentrations and temperature on the laser-induced-fluorescence (LIF) measurement of CO in turbulent flames is described, under conditions in which the fluorescence and the temperature are measured independently. The analysis shows that correlations between CO concentration and temperature can bias the averaged mole fraction extracted from LIF measurements. The magnitude of the bias can exceed the order of the average CO mole fraction. Further, LIF measurements of CO concentrations in a turbulent, nonpremixed, natural gas flame are described. The averaged CO mole fractions are derived from the fluorescence measurements by the use of flame temperatures independently measured by coherent anti-Stokes Raman spectroscopy. Analysis of the fluctuations in measured temperature and fluorescence indicates that temperature and CO concentrations in flame regions with intensive mixing are indeed correlated. In the flame regions where burnout of CO has ceased, the LIF measurements of the CO mole fraction correspond to the probe measurements in exhaust.

Ex-vivo UV autofluorescence imaging and fluorescence spectroscopy of atherosclerotic pathology in human aorta

NASA Astrophysics Data System (ADS)

Lewis, William; Williams, Maura; Franco, Walfre

2017-02-01

The aim of our study was to identify fluorescence excitation-emission pairs correlated with atherosclerotic pathology in ex-vivo human aorta. Wide-field images of atherosclerotic human aorta were captured using UV and visible excitation and emission wavelength pairs of several known fluorophores to investigate correspondence with gross pathologic features. Fluorescence spectroscopy and histology were performed on 21 aortic samples. A matrix of Pearson correlation coefficients were determined for the relationship between relevant histologic features and the intensity of emission for 427 wavelength pairs. A multiple linear regression analysis indicated that elastin (370/460 nm) and tryptophan (290/340 nm) fluorescence predicted 58% of the variance in intima thickness (R-squared = 0.588, F(2,18) = 12.8, p=.0003), and 48% of the variance in media thickness (R-squared = 0.483, F(2,18) = 8.42, p=.002), suggesting that endogenous fluorescence intensity at these wavelengths can be utilized for improved pathologic characterization of atherosclerotic plaques.

Remote temperature measurements in femto-liter volumes using dual-focus-Fluorescence Correlation Spectroscopy.

PubMed

Müller, Claus B; Weiss, Kerstin; Loman, Anastasia; Enderlein, Jörg; Richtering, Walter

2009-05-07

Remote temperature measurements in microfluidic devices with micrometer spatial resolution are important for many applications in biology, biochemistry and chemistry. The most popular methods use the temperature-dependent fluorescence lifetime of Rhodamine B, or the temperature-dependent size of thermosensitive materials such as microgel particles. Here, we use the recently developed method of dual-focus fluorescence correlation spectroscopy (2fFCS) for measuring the absolute diffusion coefficient of small fluorescent molecules at nanomolar concentrations and show how these data can be used for remote temperature measurements on a micrometer scale. We perform comparative temperature measurements using all three methods and show that the accuracy of 2fFCS is comparable or even better than that achievable with Rhodamine B fluorescence lifetime measurements. The temperature dependent microgel swelling leads to an enhanced accuracy within a narrow temperature range around the volume phase transition temperature, but requires the availability of specific microgels, whereas 2fFCS is applicable under very general conditions.

Mueller matrix signature in advanced fluorescence microscopy imaging

NASA Astrophysics Data System (ADS)

Mazumder, Nirmal; Qiu, Jianjun; Kao, Fu-Jen; Diaspro, Alberto

2017-02-01

We have demonstrated the measurement and characterization of the polarization properties of a fluorescence signal using four-channel photon counting based Stokes-Mueller polarization microscopy. Thus, Lu-Chipman decomposition was applied to extract the critical polarization properties such as depolarization, linear retardance and the optical rotation of collagen type I fiber. We observed the spatial distribution of anisotropic and helical molecules of collagen from the reconstructed 2D Mueller images based on the fluorescence signal in a pixel-by-pixel manner.

Understanding the Relative Contributions of and Critical Enzymes for the Three Pathways for Intracrine Metabolism of Testicular Androgens in Advanced Prostate Cancer

DTIC Science & Technology

2017-10-01

of the project as stated in the approved SOW. If the application listed milestones/target dates for important activities or phases of the project...These fluorescent AND, 5α-dione and DIOL derivatives retained the needed level of biological activity in biological assays (vida infra). In...stably expressed AR fused with green fluorescent protein (GFP) was used to determine cellular uptake of fluorescent- androgens, AR activation and

Nondestructive assessment of collagen hydrogel cross-linking using time-resolved autofluorescence imaging.

PubMed

Sherlock, Benjamin E; Harvestine, Jenna N; Mitra, Debika; Haudenschild, Anne; Hu, Jerry; Athanasiou, Kyriacos A; Leach, J Kent; Marcu, Laura

2018-03-01

We investigate the use of a fiber-based, multispectral fluorescence lifetime imaging (FLIm) system to nondestructively monitor changes in mechanical properties of collagen hydrogels caused by controlled application of widely used cross-linking agents, glutaraldehyde (GTA) and ribose. Postcross-linking, fluorescence lifetime images are acquired prior to the hydrogels being processed by rheological or tensile testing to directly probe gel mechanical properties. To preserve the sterility of the ribose-treated gels, FLIm is performed inside a biosafety cabinet (BSC). A pairwise correlation analysis is used to quantify the relationship between mean hydrogel fluorescence lifetimes and the storage or Young's moduli of the gels. In the GTA study, we observe strong and specific correlations between fluorescence lifetime and the storage and Young's moduli. Similar correlations are not observed in the ribose study and we postulate a reason for this. Finally, we demonstrate the ability of FLIm to longitudinally monitor dynamic cross-link formation. The strength of the GTA correlations and deployment of our fiber-based FLIm system inside the aseptic environment of a BSC suggests that this technique may be a valuable tool for the tissue engineering community where longitudinal assessment of tissue construct maturation in vitro is highly desirable. (2018) COPYRIGHT Society of Photo-Optical Instrumentation Engineers (SPIE).

Sample-Averaged Biexciton Quantum Yield Measured by Solution-Phase Photon Correlation

DOE PAGES

Beyler, Andrew P.; Bischof, Thomas S.; Cui, Jian; ...

2014-11-19

The brightness of nanoscale optical materials such as semiconductor nanocrystals is currently limited in high excitation flux applications by inefficient multiexciton fluorescence. We have devised a solution-phase photon correlation measurement that can conveniently and reliably measure the average biexciton-to-exciton quantum yield ratio of an entire sample without user selection bias. This technique can be used to investigate the multiexciton recombination dynamics of a broad scope of synthetically underdeveloped materials, including those with low exciton quantum yields and poor fluorescence stability. Here in this study, we have applied this method to measure weak biexciton fluorescence in samples of visible-emitting InP/ZnS andmore » InAs/ZnS core/shell nanocrystals, and to demonstrate that a rapid CdS shell growth procedure can markedly increase the biexciton fluorescence of CdSe nanocrystals.« less

Monitoring of an antigen manufacturing process.

PubMed

Zavatti, Vanessa; Budman, Hector; Legge, Raymond; Tamer, Melih

2016-06-01

Fluorescence spectroscopy in combination with multivariate statistical methods was employed as a tool for monitoring the manufacturing process of pertactin (PRN), one of the virulence factors of Bordetella pertussis utilized in whopping cough vaccines. Fluorophores such as amino acids and co-enzymes were detected throughout the process. The fluorescence data collected at different stages of the fermentation and purification process were treated employing principal component analysis (PCA). Through PCA, it was feasible to identify sources of variability in PRN production. Then, partial least square (PLS) was employed to correlate the fluorescence spectra obtained from pure PRN samples and the final protein content measured by a Kjeldahl test from these samples. In view that a statistically significant correlation was found between fluorescence and PRN levels, this approach could be further used as a method to predict the final protein content.

Sample-Averaged Biexciton Quantum Yield Measured by Solution-Phase Photon Correlation

PubMed Central

Beyler, Andrew P.; Bischof, Thomas S.; Cui, Jian; Coropceanu, Igor; Harris, Daniel K.; Bawendi, Moungi G.

2015-01-01

The brightness of nanoscale optical materials such as semiconductor nanocrystals is currently limited in high excitation flux applications by inefficient multiexciton fluorescence. We have devised a solution-phase photon correlation measurement that can conveniently and reliably measure the average biexciton-to-exciton quantum yield ratio of an entire sample without user selection bias. This technique can be used to investigate the multiexciton recombination dynamics of a broad scope of synthetically underdeveloped materials, including those with low exciton quantum yields and poor fluorescence stability. Here, we have applied this method to measure weak biexciton fluorescence in samples of visible-emitting InP/ZnS and InAs/ZnS core/shell nanocrystals, and to demonstrate that a rapid CdS shell growth procedure can markedly increase the biexciton fluorescence of CdSe nanocrystals. PMID:25409496

Laser-induced fluorescence ratios of Cajanus cajan L. under the stress of cadmium and its correlation with pigment content and pigment ratios.

PubMed

Maurya, Renu; Gopal, R

2008-04-01

Laser-induced fluorescence spectra were used to characterize the effect of cadmium on the pigment status of the leaves of Cajanus cajan L. Laser-induced fluorescence spectra of untreated as well as cadmium treated (0.01 mM, 0.10 mM, and 1.00 mM) Cajanus cajan L. were recorded using the 355 nm line of a Nd:YAG laser as the excitation source and a monochromator with an intensified charge-coupled device as a detector in the region 400-800 nm. The fluorescence intensity ratios (FIR) of control as well as treated Cajanus cajan L. have been calculated by evaluating curve fitted parameters using a Gaussian spectral function. In addition, some growth parameters, such as photosynthetic pigment content, were also measured. The 355 nm line of the laser-light-excited leaves not only showed a fluorescence emission in the red spectral region (650-800 nm), but also in the blue-green region (400-570 nm). The chlorophyll FIR F690/F740 strongly correlated with the photosynthetic pigment content (total chlorophyll and carotenoids) and its ratio. Consequently, a correlation was also seen between the ratio of the blue-green fluorescence F470/F540 and the photosynthetic pigment content. The results indicated that the plants treated with 0.01 mM of cadmium exhibited better growth, while higher concentrations of cadmium were hazardous for Cajanus cajan L.

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.

Organic fluorescent dye-based nanomaterials: Advances in the rational design for imaging and sensing applications.

PubMed

Svechkarev, Denis; Mohs, Aaron M

2018-02-25

Self-assembled fluorescent nanomaterials based on small-molecule organic dyes are gaining increasing popularity in imaging and sensing applications over the past decade. This is primarily due to their ability to combine spectral property tunability and biocompatibility of small molecule organic fluorophores with brightness, chemical, and colloidal stability of inorganic materials. Such a unique combination of features comes with rich versatility of dye-based nanomaterials: from aggregates of small molecules to sophisticated core-shell nanoarchitectures involving hyperbranched polymers. Along with the ongoing discovery of new materials and better ways of their synthesis, it is very important to continue systematic studies of fundamental factors that regulate the key properties of fluorescent nanomaterials: their size, polydispersity, colloidal stability, chemical stability, absorption and emission maxima, biocompatibility, and interactions with biological interfaces. In this review, we focus on the systematic description of various types of organic fluorescent nanomaterials, approaches to their synthesis, and ways to optimize and control their characteristics. The discussion is built on examples from reports on recent advances in design and applications of such materials. Conclusions made from this analysis allow a perspective on future development of fluorescent nanomaterials design for biomedical and related applications. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Zinc Oxide Nanomaterials for Biomedical Fluorescence Detection

PubMed Central

Hahm, Jong-in

2014-01-01

One-dimensional zinc oxide nanomaterials have been recently developed into novel, extremely effective, optical signal-enhancing bioplatforms. Their usefulness has been demonstrated in various biomedical fluorescence assays. Fluorescence is extensively used in biology and medicine as a sensitive and noninvasive detection method for tracking and analyzing biological molecules. Achieving high sensitivity via improving signal-to-noise ratio is of paramount importance in fluorescence-based, trace-level detection. Recent advances in the development of optically superior one-dimensional materials have contributed to this important biomedical area of detection. This review article will discuss major research developments that have so far been made in this emerging and exciting topical field. The discussion will cover a broad range of subjects including synthesis of zinc oxide nanorods (ZnO NRs), various properties differentiating them as suitable optical biodetection platforms, their demonstrated applicability in DNA and protein detection, and the nanomaterial characteristics relevant for biomolecular fluorescence enhancement. This review will then summarize the current status of ZnO NR-based biodetection and further elaborate future utility of ZnO NR platforms for advanced biomedical assays, based on their proven advantages. Lastly, present challenges experienced in this topical area will be identified and focal subject areas for future research will be suggested as well. PMID:24730276

Spitzenkörper Localization and Intracellular Traffic of Green Fluorescent Protein-Labeled CHS-3 and CHS-6 Chitin Synthases in Living Hyphae of Neurospora crassa▿ †

PubMed Central

Riquelme, Meritxell; Bartnicki-García, Salomon; González-Prieto, Juan Manuel; Sánchez-León, Eddy; Verdín-Ramos, Jorge A.; Beltrán-Aguilar, Alejandro; Freitag, Michael

2007-01-01

The subcellular location and traffic of two selected chitin synthases (CHS) from Neurospora crassa, CHS-3 and CHS-6, labeled with green fluorescent protein (GFP), were studied by high-resolution confocal laser scanning microscopy. While we found some differences in the overall distribution patterns and appearances of CHS-3-GFP and CHS-6-GFP, most features were similar and were observed consistently. At the hyphal apex, fluorescence congregated into a conspicuous single body corresponding to the location of the Spitzenkörper (Spk). In distal regions (beyond 40 μm from the apex), CHS-GFP revealed a network of large endomembranous compartments that was predominantly comprised of irregular tubular shapes, while some compartments were distinctly spherical. In the distal subapex (20 to 40 μm from the apex), fluorescence was observed in globular bodies that appeared to disintegrate into vesicles as they advanced forward until reaching the proximal subapex (5 to 20 μm from the apex). CHS-GFP was also conspicuously found delineating developing septa. Analysis of fluorescence recovery after photobleaching suggested that the fluorescence of the Spk originated from the advancing population of microvesicles (chitosomes) in the subapex. The inability of brefeldin A to interfere with the traffic of CHS-containing microvesicles and the lack of colocalization of CHS-GFP with the endoplasmic reticulum (ER)-Golgi body fluorescent dyes lend support to the idea that CHS proteins are delivered to the cell surface via an alternative route distinct from the classical ER-Golgi body secretory pathway. PMID:17644657

A portable system for noninvasive assessment of advanced glycation end-products using skin fluorescence and reflectance spectrum

NASA Astrophysics Data System (ADS)

Wang, Y. K.; Zhu, L.; Zhang, L.; Zhang, G.; Liu, Y.; Wang, A.

2012-07-01

An optical system has been developed for noninvasive assessment of skin advanced glycation end-products (AGEs). The system comprises mainly a high-power ultraviolet light emitting diode (LED) as an excitation source, an LED array for the reflectance measurement, a trifurcated fiber-optic probe for light transmitting and receiving, and a compact spectrometer for light detecting. Both skin fluorescence of a subject and the reflectance spectrum of the same site can be obtained in a single measurement with the system. Demonstrative measurements with the system have been conducted. Results indicate that the measured reflectance spectrum can be used to compensate for the distortion of AGEs fluorescence, which is caused by skin absorption and scattering. The system is noninvasive, portable, easy to operate, and has potential applications for clinical diagnosis of AGE-related diseases, especially diabetes mellitus.

A Simple, Low-Cost Platform for Real-Time Isothermal Nucleic Acid Amplification

PubMed Central

Craw, Pascal; Mackay, Ruth E.; Naveenathayalan, Angel; Hudson, Chris; Branavan, Manoharanehru; Sadiq, S. Tariq; Balachandran, Wamadeva

2015-01-01

Advances in microfluidics and the introduction of isothermal nucleic acid amplification assays have resulted in a range of solutions for nucleic acid amplification tests suited for point of care and field use. However, miniaturisation of instrumentation for such assays has not seen such rapid advances and fluorescence based assays still depend on complex, bulky and expensive optics such as fluorescence microscopes, photomultiplier tubes and sensitive lens assemblies. In this work we demonstrate a robust, low cost platform for isothermal nucleic acid amplification on a microfluidic device. Using easily obtainable materials and commercial off-the-shelf components, we show real time fluorescence detection using a low cost photodiode and operational amplifier without need for lenses. Temperature regulation on the device is achieved using a heater fabricated with standard printed circuit board fabrication methods. These facile construction methods allow fabrications at a cost compatible with widespread deployment to resource poor settings. PMID:26389913

Functional Carbon Quantum Dots: A Versatile Platform for Chemosensing and Biosensing.

PubMed

Feng, Hui; Qian, Zhaosheng

2018-05-01

Carbon quantum dot has emerged as a new promising fluorescent nanomaterial due to its excellent optical properties, outstanding biocompatibility and accessible fabrication methods, and has shown huge application perspective in a variety of areas, especially in chemosensing and biosensing applications. In this personal account, we give a brief overview of carbon quantum dots from its origin and preparation methods, present some advance on fluorescence origin of carbon quantum dots, and focus on development of chemosensors and biosensors based on functional carbon quantum dots. Comprehensive advances on functional carbon quantum dots as a versatile platform for sensing from our group are included and summarized as well as some typical examples from the other groups. The biosensing applications of functional carbon quantum dots are highlighted from selective assays of enzyme activity to fluorescent identification of cancer cells and bacteria. © 2018 The Chemical Society of Japan & Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Evaluation of CDOM sources and their links with water quality in the lakes of Northeast China using fluorescence spectroscopy

NASA Astrophysics Data System (ADS)

Zhao, Ying; Song, Kaishan; Wen, Zhidan; Fang, Chong; Shang, Yingxin; Lv, Lili

2017-07-01

The spatial distributions of the fluorescence intensities Fmax for chromophoric dissolved organic matter (CDOM) components, the fluorescence indices (FI370 and FI310) and their correlations with water quality of 19 lakes in the Songhua River Basin (SHRB) across semiarid regions of Northeast China were examined with the data collected in September 2012 and 2015. The 19 lakes were divided into two groups according to EC (threshold value = 800 μS cm-1): fresh water (N = 13) and brackish water lakes (N = 6). The fluorescent characteristics of CDOM in the 19 lakes were investigated using excitation-emission matrix fluorescence spectroscopy (EEM) coupled with parallel factor (PARAFAC) and multivariate analysis. Two humic-like components (C1 and C3), one tryptophan-like component (C2), and one tyrosine-like component (C4) were identified by PARAFAC. The component C4 was not included in subsequent analyses due to the strong scatter in some colloidal water samples from brackish water lakes. The correlations between Fmax for the three EEM-PARAFAC extracted CDOM components C1-C3, the fluorescence indices (FI370 and FI310) and the water quality parameters (i.e., TN, TP, Chl-a, pH, EC, turbidity (Turb) and dissolved organic carbon (DOC)) were determined by redundancy analysis (RDA). The results of RDA analysis showed that spatial variation in land cover, pollution sources, and salinity/EC gradients in water quality affected Fmax for the fluorescent components C1-C3 and the fluorescence indices (FI370 and FI310). Further examination indicated that the CDOM fluorescent components and the fluorescence indices (FI370 and FI310) did not significantly differ (t-test, p > 0.05) in fresh water (N = 13) and brackish water lakes (N = 6). There was a difference in the distribution of the average Fmax for the CDOM fluorescent components between C1 to C3 from agricultural sources and urban wastewater sources in hypereutrophic brackish water lakes. The Fmax for humic-like components C1 and C3 spatially varied with land cover among the 19 lakes. Our results indicated that the spatial distributions of Fmax for CDOM fluorescent components and their correlations with water quality can be evaluated by EEM-PARAFAC and multivariate analysis among the 19 lakes across semiarid regions of Northeast China, which has potential implication for lakes with similar genesis.

A label-free and enzyme-free system for operating various logic devices using poly(thymine)-templated CuNPs and SYBR Green I as signal transducers

NASA Astrophysics Data System (ADS)

Wu, Changtong; Zhou, Chunyang; Wang, Erkang; Dong, Shaojun

2016-07-01

For the first time by integrating fluorescent polyT-templated CuNPs and SYBR Green I, a basic INHIBIT gate and four advanced logic circuits (2-to-1 encoder, 4-to-2 encoder, 1-to-2 decoder and 1-to-2 demultiplexer) have been conceptually realized under label-free and enzyme-free conditions. Taking advantage of the selective formation of CuNPs on ss-DNA, the implementation of these advanced logic devices were achieved without any usage of dye quenching groups or other nanomaterials like graphene oxide or AuNPs since polyA strands not only worked as an input but also acted as effective inhibitors towards polyT templates, meeting the aim of developing bio-computing with cost-effective and operationally simple methods. In short, polyT-templated CuNPs, as promising fluorescent signal reporters, are successfully applied to fabricate advanced logic devices, which may present a potential path for future development of molecular computations.For the first time by integrating fluorescent polyT-templated CuNPs and SYBR Green I, a basic INHIBIT gate and four advanced logic circuits (2-to-1 encoder, 4-to-2 encoder, 1-to-2 decoder and 1-to-2 demultiplexer) have been conceptually realized under label-free and enzyme-free conditions. Taking advantage of the selective formation of CuNPs on ss-DNA, the implementation of these advanced logic devices were achieved without any usage of dye quenching groups or other nanomaterials like graphene oxide or AuNPs since polyA strands not only worked as an input but also acted as effective inhibitors towards polyT templates, meeting the aim of developing bio-computing with cost-effective and operationally simple methods. In short, polyT-templated CuNPs, as promising fluorescent signal reporters, are successfully applied to fabricate advanced logic devices, which may present a potential path for future development of molecular computations. Electronic supplementary information (ESI) available. See DOI: 10.1039/c6nr04069a

Magneto-Fluorescent Core-Shell Supernanoparticles

PubMed Central

Chen, Ou; Riedemann, Lars; Etoc, Fred; Herrmann, Hendrik; Coppey, Mathieu; Barch, Mariya; Farrar, Christian T.; Zhao, Jing; Bruns, Oliver T.; Wei, He; Guo, Peng; Cui, Jian; Jensen, Russ; Chen, Yue; Harris, Daniel K.; Cordero, Jose M.; Wang, Zhongwu; Jasanoff, Alan; Fukumura, Dai; Reimer, Rudolph; Dahan, Maxime; Jain, Rakesh K.; Bawendi, Moungi G.

2014-01-01

Magneto-fluorescent particles have been recognized as an emerging class of materials that exhibit great potential in advanced applications. However, synthesizing such magneto-fluorescent nanomaterials that simultaneously exhibit uniform and tunable sizes, high magnetic content loading, maximized fluorophore coverage at the surface, and a versatile surface functionality has proven challenging. Here we report a simple approach for co-assembling magnetic nanoparticles with fluorescent quantum dots to form colloidal magneto-fluorescent supernanoparticles. Importantly, these supernanoparticles exhibit a superstructure consisting of a close packed magnetic nanoparticle “core” which is fully surrounded by a “shell” of fluorescent quantum dots. A thin layer of silica-coating provides high colloidal stability and biocompatiblity and a versatile surface functionality. We demonstrate that after surface pegylation, these silica-coated magneto-fluorescent supernanoparticles can be magnetically manipulated inside living cells while being optically tracked. Moreover, our silica-coated magneto-fluorescent supernanoparticles can also serve as an in vivo multi-photon and magnetic resonance dual-modal imaging probe. PMID:25298155

Fluorescence microscopy: A tool to study autophagy

NASA Astrophysics Data System (ADS)

Rai, Shashank; Manjithaya, Ravi

2015-08-01

Autophagy is a cellular recycling process through which a cell degrades old and damaged cellular components such as organelles and proteins and the degradation products are reused to provide energy and building blocks. Dysfunctional autophagy is reported in several pathological situations. Hence, autophagy plays an important role in both cellular homeostasis and diseased conditions. Autophagy can be studied through various techniques including fluorescence based microscopy. With the advancements of newer technologies in fluorescence microscopy, several novel processes of autophagy have been discovered which makes it an essential tool for autophagy research. Moreover, ability to tag fluorescent proteins with sub cellular targets has enabled us to evaluate autophagy processes in real time under fluorescent microscope. In this article, we demonstrate different aspects of autophagy in two different model organisms i.e. yeast and mammalian cells, with the help of fluorescence microscopy.

Confocal mosaicing microscopy of human skin ex vivo: spectral analysis for digital staining to simulate histology-like appearance

NASA Astrophysics Data System (ADS)

Bini, Jason; Spain, James; Nehal, Kishwer; Hazelwood, Vikki; Dimarzio, Charles; Rajadhyaksha, Milind

2011-07-01

Confocal mosaicing microscopy enables rapid imaging of large areas of fresh tissue, without the processing that is necessary for conventional histology. Mosaicing may offer a means to perform rapid histology at the bedside. A possible barrier toward clinical acceptance is that the mosaics are based on a single mode of grayscale contrast and appear black and white, whereas histology is based on two stains (hematoxylin for nuclei, eosin for cellular cytoplasm and dermis) and appears purple and pink. Toward addressing this barrier, we report advances in digital staining: fluorescence mosaics that show only nuclei, are digitally stained purple and overlaid on reflectance mosaics, which show only cellular cytoplasm and dermis, and are digitally stained pink. With digital staining, the appearance of confocal mosaics mimics the appearance of histology. Using multispectral analysis and color matching functions, red, green, and blue (RGB) components of hematoxylin and eosin stains in tissue were determined. The resulting RGB components were then applied in a linear algorithm to transform fluorescence and reflectance contrast in confocal mosaics to the absorbance contrast seen in pathology. Optimization of staining with acridine orange showed improved quality of digitally stained mosaics, with good correlation to the corresponding histology.

Fast fluorescence techniques for crystallography beamlines

PubMed Central

Stepanov, Sergey; Hilgart, Mark; Yoder, Derek W.; Makarov, Oleg; Becker, Michael; Sanishvili, Ruslan; Ogata, Craig M.; Venugopalan, Nagarajan; Aragão, David; Caffrey, Martin; Smith, Janet L.; Fischetti, Robert F.

2011-01-01

This paper reports on several developments of X-ray fluorescence techniques for macromolecular crystallography recently implemented at the National Institute of General Medical Sciences and National Cancer Institute beamlines at the Advanced Photon Source. These include (i) three-band on-the-fly energy scanning around absorption edges with adaptive positioning of the fine-step band calculated from a coarse pass; (ii) on-the-fly X-ray fluorescence rastering over rectangular domains for locating small and invisible crystals with a shuttle-scanning option for increased speed; (iii) fluorescence rastering over user-specified multi-segmented polygons; and (iv) automatic signal optimization for reduced radiation damage of samples. PMID:21808424

The X-ray Fluorescence Microscopy Beamline at the Australian Synchrotron

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

Paterson, D.; Jonge, M. D. de; Howard, D. L.

2011-09-09

A hard x-ray micro-nanoprobe has commenced operation at the Australian Synchrotron providing versatile x-ray fluorescence microscopy across an incident energy range from 4 to 25 keV. Two x-ray probes are used to collect {mu}-XRF and {mu}-XANES for elemental and chemical microanalysis: a Kirkpatrick-Baez mirror microprobe for micron resolution studies and a Fresnel zone plate nanoprobe capable of 60-nm resolution. Some unique aspects of the beamline design and operation are discussed. An advanced energy dispersive x-ray fluorescence detection scheme named Maia has been developed for the beamline, which enables ultrafast x-ray fluorescence microscopy.

Azadioxatriangulenium: a long fluorescence lifetime fluorophore for large biomolecule binding assay

NASA Astrophysics Data System (ADS)

Just Sørensen, Thomas; Thyrhaug, Erling; Szabelski, Mariusz; Luchowski, Rafal; Gryczynski, Ignacy; Gryczynski, Zygmunt; Laursen, Bo W.

2013-06-01

Of the many optical bioassays available, sensing by fluorescence anisotropy has great advantages as it provides a sensitive, instrumentally simple, ratiometric method of detection. However, it is hampered by a severe limitation, as the emission lifetime of the label needs to be comparable to the correlation lifetime (tumbling time) of the biomolecule which is labelled. For proteins of moderate size this is on the order of 20-200 ns, which due to practical issues currently limits the choice of labels to the dansyl-type dyes and certain aromatic dyes. These have the significant drawback of UV/blue absorption and emission as well as an often significant solvent sensitivity. Here, we report the synthesis and characterization of a new fluorescent label for high molecular weight biomolecule assay based on the azadioxatriangulenium motif. The NHS ester of the long fluorescence lifetime, red-emitting fluorophore: azadioxatriangulenium (ADOTA-NHS) was conjugated to anti-rabbit Immunoglobulin G (antiIgG). The long fluorescence lifetime was exploited to determine the correlation time of the high molecular weight antibody and its complex with rabbit Immunoglobulin G (IgG) with steady-state fluorescence anisotropy and time-resolved methods: solution phase immuno-assay was performed following either steady-state or time-resolved fluorescence anisotropy. By performing a variable temperature experiment it was determined that the binding of the ligand resulted in an increase in correlation time of more than 75%, and an increase in the steady-state anisotropy of 18%. The results show that the triangulenium class of dyes can be used in anisotropy assay to detect binding events involving biomolecules of far larger size than what is possible with most other red-emitting organic dyes.

Simultaneous AFM and fluorescence imaging: A method for aligning an AFM-tip with an excitation beam using a 2D galvanometer

NASA Astrophysics Data System (ADS)

Moores, A. N.; Cadby, A. J.

2018-02-01

Correlative fluorescence and atomic force microscopy (AFM) imaging is a highly attractive technique for use in biological imaging, enabling force and mechanical measurements of particular structures whose locations are known due to the specificity of fluorescence imaging. The ability to perform these two measurements simultaneously (rather than consecutively with post-processing correlation) is highly valuable because it would allow the mechanical properties of a structure to be tracked over time as changes in the sample occur. We present an instrument which allows simultaneous AFM and fluorescence imaging by aligning an incident fluorescence excitation beam with an AFM-tip. Alignment was performed by calibrating a 2D galvanometer present in the excitation beam path and using it to reposition the incident beam. Two programs were developed (one manual and one automated) which correlate sample features between the AFM and fluorescence images, calculating the distance required to translate the incident beam towards the AFM-tip. Using this method, we were able to obtain beam-tip alignment (and therefore field-of-view alignment) from an offset of >15 μm to within one micron in two iterations of the program. With the program running alongside data acquisition for real-time feedback between AFM and optical images, this offset was maintained over a time period of several hours. Not only does this eliminate the need to image large areas with both techniques to ensure that fields-of-view overlap, but it also raises the possibility of using this instrument for tip-enhanced fluorescence applications, a technique in which super-resolution images have previously been achieved.

Fluorescence imaging of tryptophan and collagen cross-links to evaluate wound closure ex vivo

NASA Astrophysics Data System (ADS)

Wang, Ying; Ortega-Martinez, Antonio; Farinelli, Bill; Anderson, R. R.; Franco, Walfre

2016-02-01

Wound size is a key parameter in monitoring healing. Current methods to measure wound size are often subjective, time-consuming and marginally invasive. Recently, we developed a non-invasive, non-contact, fast and simple but robust fluorescence imaging (u-FEI) method to monitor the healing of skin wounds. This method exploits the fluorescence of native molecules to tissue as functional and structural markers. The objective of the present study is to demonstrate the feasibility of using variations in the fluorescence intensity of tryptophan and cross-links of collagen to evaluate proliferation of keratinocyte cells and quantitate size of wound during healing, respectively. Circular dermal wounds were created in ex vivo human skin and cultured in different media. Two serial fluorescence images of tryptophan and collagen cross-links were acquired every two days. Histology and immunohistology were used to validate correlation between fluorescence and epithelialization. Images of collagen cross-links show fluorescence of the exposed dermis and, hence, are a measure of wound area. Images of tryptophan show higher fluorescence intensity of proliferating keratinocytes forming new epithelium, as compared to surrounding keratinocytes not involved in epithelialization. These images are complementary since collagen cross-links report on structure while tryptophan reports on function. HE and immunohistology show that tryptophan fluorescence correlates with newly formed epidermis. We have established a fluorescence imaging method for studying epithelialization processes during wound healing in a skin organ culture model, our approach has the potential to provide a non-invasive, non-contact, quick, objective and direct method for quantitative measurements in wound healing in vivo.

The Effect of a Fluorophore Photo-Physics on the Lipid Vesicle Diffusion Coefficient Studied by Fluorescence Correlation Spectroscopy.

PubMed

Drabik, Dominik; Przybyło, Magda; Sikorski, Aleksander; Langner, Marek

2016-03-01

Fluorescence Correlation Spectroscopy (FCS) is a technique, which allows determination of the diffusion coefficient and concentration of fluorescent objects suspended in the solution. The measured parameter is the fluctuation of the fluorescence signal emitted by diffusing molecules. When 100 nm DOPC vesicles labeled with various fluorescent dyes (Fluorescein-PE, NBD-PE, Atto488 DOPE or βBodipy FL) were measured, different values of diffusion coefficients have been obtained. These diffusion coefficients were different from the expected values measured using the dynamic light scattering method (DLS). The FCS was initially developed for solutions containing small fluorescent molecules therefore the observed inconsistency may result from the nature of vesicle suspension itself. The duration of the fluorescence signal may depend on the following factors: the exposure time of the labeled object to the excitation beam, the photo-physical properties (e.g., stability) of a fluorophore, the theoretical model used for the calculations of the diffusion coefficient and optical properties of the vesicle suspension. The diffusion coefficients determined for differently labeled liposomes show that its dependence on vesicle size and quantity of fluorescent probed used for labeling was significant demonstrating that the fluorescence properties of the fluorophore itself (bleaching and/or blinking) were critical factors for a correct outcome of FCS experiment. The new, based on combined FCS and DLS measurements, method for the determination of the focal volume prove itself to be useful for the evaluation of a fluorescence dye with respect to its applicability for FCS experiment.

Depth-resolved imaging of colon tumor using optical coherence tomography and fluorescence laminar optical tomography (Conference Presentation)

NASA Astrophysics Data System (ADS)

Tang, Qinggong; Frank, Aaron; Wang, Jianting; Chen, Chao-wei; Jin, Lily; Lin, Jon; Chan, Joanne M.; Chen, Yu

2016-03-01

Early detection of neoplastic changes remains a critical challenge in clinical cancer diagnosis and treatment. Many cancers arise from epithelial layers such as those of the gastrointestinal (GI) tract. Current standard endoscopic technology is unable to detect those subsurface lesions. Since cancer development is associated with both morphological and molecular alterations, imaging technologies that can quantitative image tissue's morphological and molecular biomarkers and assess the depth extent of a lesion in real time, without the need for tissue excision, would be a major advance in GI cancer diagnostics and therapy. In this research, we investigated the feasibility of multi-modal optical imaging including high-resolution optical coherence tomography (OCT) and depth-resolved high-sensitivity fluorescence laminar optical tomography (FLOT) for structural and molecular imaging. APC (adenomatous polyposis coli) mice model were imaged using OCT and FLOT and the correlated histopathological diagnosis was obtained. Quantitative structural (the scattering coefficient) and molecular imaging parameters (fluorescence intensity) from OCT and FLOT images were developed for multi-parametric analysis. This multi-modal imaging method has demonstrated the feasibility for more accurate diagnosis with 87.4% (87.3%) for sensitivity (specificity) which gives the most optimal diagnosis (the largest area under receiver operating characteristic (ROC) curve). This project results in a new non-invasive multi-modal imaging platform for improved GI cancer detection, which is expected to have a major impact on detection, diagnosis, and characterization of GI cancers, as well as a wide range of epithelial cancers.

Advanced Diagnostics for Reacting Flows

DTIC Science & Technology

2006-06-01

TECHNICAL DISCUSSION: 1. Infrared-PLIF Imaging Diagnostics using Vibrational Transitions IR-PLIF allows for imaging a group of molecular species important...excitation of IR-active vibrational modes with imaging of the subsequent vibrational fluorescence. Quantitative interpretation requires knowledge of...the vibrational energy transfer processes, and hence in recent years we have been developing models for infrared fluorescence. During the past year

Synthesis and Application of Ratiometric and "Turn-On" Fluorescent pH Sensors: An Advanced Organic Undergraduate Laboratory

ERIC Educational Resources Information Center

Hutt, Johnathon T.; Aron, Zachary D.

2014-01-01

An upper-division organic chemistry laboratory experiment exploring fluorescent sensing over two laboratory periods and part of a third is described. Two functionally distinct pH-responsive sensors are prepared through a dehydrative three-component coupling reaction. During the abbreviated (<1 h) first laboratory period, students set up…

Nitroxide amide-BODIPY probe behavior in fibroblasts analyzed by advanced fluorescence microscopy.

PubMed

Liras, M; Simoncelli, S; Rivas-Aravena, A; García, O; Scaiano, J C; Alarcon, E I; Aspée, A

2016-04-26

A novel synthesized nitroxide amide-BODIPY prefluorescent probe was used to study cellular redox balance that modulates nitroxide/hydroxylamine ratio in cultured human fibroblasts. FLIM quantitatively differentiated between nitroxide states of the cytoplasm-localized probe imaged by TIRF, monitoring nitroxide depletion by hydrogen peroxide; eluding incorrect interpretation if only fluorescence intensity is considered.

Microsecond protein dynamics observed at the single-molecule level

NASA Astrophysics Data System (ADS)

Otosu, Takuhiro; Ishii, Kunihiko; Tahara, Tahei

2015-07-01

How polypeptide chains acquire specific conformations to realize unique biological functions is a central problem of protein science. Single-molecule spectroscopy, combined with fluorescence resonance energy transfer, is utilized to study the conformational heterogeneity and the state-to-state transition dynamics of proteins on the submillisecond to second timescales. However, observation of the dynamics on the microsecond timescale is still very challenging. This timescale is important because the elementary processes of protein dynamics take place and direct comparison between experiment and simulation is possible. Here we report a new single-molecule technique to reveal the microsecond structural dynamics of proteins through correlation of the fluorescence lifetime. This method, two-dimensional fluorescence lifetime correlation spectroscopy, is applied to clarify the conformational dynamics of cytochrome c. Three conformational ensembles and the microsecond transitions in each ensemble are indicated from the correlation signal, demonstrating the importance of quantifying microsecond dynamics of proteins on the folding free energy landscape.

Microsecond protein dynamics observed at the single-molecule level

PubMed Central

Otosu, Takuhiro; Ishii, Kunihiko; Tahara, Tahei

2015-01-01

How polypeptide chains acquire specific conformations to realize unique biological functions is a central problem of protein science. Single-molecule spectroscopy, combined with fluorescence resonance energy transfer, is utilized to study the conformational heterogeneity and the state-to-state transition dynamics of proteins on the submillisecond to second timescales. However, observation of the dynamics on the microsecond timescale is still very challenging. This timescale is important because the elementary processes of protein dynamics take place and direct comparison between experiment and simulation is possible. Here we report a new single-molecule technique to reveal the microsecond structural dynamics of proteins through correlation of the fluorescence lifetime. This method, two-dimensional fluorescence lifetime correlation spectroscopy, is applied to clarify the conformational dynamics of cytochrome c. Three conformational ensembles and the microsecond transitions in each ensemble are indicated from the correlation signal, demonstrating the importance of quantifying microsecond dynamics of proteins on the folding free energy landscape. PMID:26151767

Highly stable lipid-encapsulation of fluorescent nanodiamonds for bioimaging applications.

PubMed

Sotoma, Shingo; Hsieh, Feng-Jen; Chen, Yen-Wei; Tsai, Pei-Chang; Chang, Huan-Cheng

2018-01-23

Highly stable lipid-encapsulated fluorescent nanodiamonds (FNDs) are produced by photo-crosslinking of diacetylene-containing lipids physically attached to the FND surface. Not only is this coating method simple and fast, but also it gives the FND-lipid hybrids favorable properties for bioapplications. The hybrids are useful as fluorescent biolabels as well as fiducial markers for correlative light and electron microscopy.

Nanodiamonds as multi-purpose labels for microscopy.

PubMed

Hemelaar, S R; de Boer, P; Chipaux, M; Zuidema, W; Hamoh, T; Martinez, F Perona; Nagl, A; Hoogenboom, J P; Giepmans, B N G; Schirhagl, R

2017-04-07

Nanodiamonds containing fluorescent nitrogen-vacancy centers are increasingly attracting interest for use as a probe in biological microscopy. This interest stems from (i) strong resistance to photobleaching allowing prolonged fluorescence observation times; (ii) the possibility to excite fluorescence using a focused electron beam (cathodoluminescence; CL) for high-resolution localization; and (iii) the potential use for nanoscale sensing. For all these schemes, the development of versatile molecular labeling using relatively small diamonds is essential. Here, we show the direct targeting of a biological molecule with nanodiamonds as small as 70 nm using a streptavidin conjugation and standard antibody labelling approach. We also show internalization of 40 nm sized nanodiamonds. The fluorescence from the nanodiamonds survives osmium-fixation and plastic embedding making them suited for correlative light and electron microscopy. We show that CL can be observed from epon-embedded nanodiamonds, while surface-exposed nanoparticles also stand out in secondary electron (SE) signal due to the exceptionally high diamond SE yield. Finally, we demonstrate the magnetic read-out using fluorescence from diamonds prior to embedding. Thus, our results firmly establish nanodiamonds containing nitrogen-vacancy centers as unique, versatile probes for combining and correlating different types of microscopy, from fluorescence imaging and magnetometry to ultrastructural investigation using electron microscopy.

Fluorescence Guided PDT for Optimization of Skin Cancer Treatment

NASA Astrophysics Data System (ADS)

Blanco, Kate; Moriyama, Lilian; Inada, Natalia; Kurachi, Cristina; Salvio, Ana; Leite, Everson; Menezes, Priscila; Bagnato, Vanderlei

2015-04-01

The photodynamic therapy (PDT) is an alternative technique that can be indicated for superficial basal cell carcinoma (sBCC), Bowen’s disease and actinic keratosis with high efficiency. The objective of this study is to present the importance of fluorescence imaging for PDT guidance and monitoring in real time. Confirming that the lesion is well prepared and the photosensitizer shows a homogenous distribution, the outcome after few PDT sessions will be positive and the recurrence should be lower. Our proposition in this study is use the widefield fluorescence imaging to evaluate the PDT protocol in situ and in real time for each lesion. This evaluation procedure is performed in two steps: first with the monitoring of the production of protoporphyrin IX (PpIX) induced by methyl aminolevulinate (MAL), an derivative of 5-aminolevulinic acid (ALA) and second with the detection of PpIX photobleaching after illumination. The fluorescence images provide information correlated with distinct clinical features and with the treatment outcome. Eight BCC lesions are presented and discussed in this study. Different fluorescence patterns of PpIX production and photobleaching could be correlated with the treatment response. The presented results show the potential of using widefield fluorescence imaging as a guidance tool to customized PDT.

Air pollution source identification

NASA Technical Reports Server (NTRS)

Fordyce, J. S.

1975-01-01

The techniques available for source identification are reviewed: remote sensing, injected tracers, and pollutants themselves as tracers. The use of the large number of trace elements in the ambient airborne particulate matter as a practical means of identifying sources is discussed. Trace constituents are determined by sensitive, inexpensive, nondestructive, multielement analytical methods such as instrumental neutron activation and charged particle X-ray fluorescence. The application to a large data set of pairwise correlation, the more advanced pattern recognition-cluster analysis approach with and without training sets, enrichment factors, and pollutant concentration rose displays for each element is described. It is shown that elemental constituents are related to specific source types: earth crustal, automotive, metallurgical, and more specific industries. A field-ready source identification system based on time and wind direction resolved sampling is described.

Serum levels of advanced glycation end-products (AGEs) and the decoy soluble receptor for AGEs (sRAGE) can identify non-alcoholic fatty liver disease in age-, sex- and BMI-matched normo-glycemic adults.

PubMed

Palma-Duran, Susana A; Kontogianni, Meropi D; Vlassopoulos, Antonis; Zhao, Shudong; Margariti, Aikaterini; Georgoulis, Michael; Papatheodoridis, George; Combet, Emilie

2018-06-01

Non-alcoholic fatty liver disease (NAFLD) is a serious health problem affecting ~25% of the global population. While NAFLD pathogenesis is still unclear, multiple NAFLD parameters, including reduced insulin sensitivity, impaired glucose metabolism and increased oxidative stress are hypothesised to foster the formation of advanced glycation end-products (AGEs). Given the link of AGEs with end organ damage, there is scope to examine the role of the AGE/RAGE axis activation in liver injury and NAFLD. Age, sex and body mass index matched normo-glycemic NAFLD adults (n = 58) and healthy controls (n = 58) were enrolled in the study. AGEs were analysed by liquid chromatography-mass spectrometry (CML, CEL), fluorescence (pentosidine, AGE fluorescence), colorimetry (fructosamine) and ELISA (sRAGE). Their association with liver function, inflammation, fibrosis and stage of NAFLD was examined. Early and advanced glycation end-products, except N ε -carboxymethyl-L-lysine (CML), were 10-30% higher, sRAGE levels 1.7-fold lower, and glycation/sRAGE ratios 4-fold higher in the NAFLD cases compared to controls. While AGEs presented weak to moderate correlations with indices of liver function and damage (AST/ALT, HOMA-IR, TNF-α and TGF-β1), including sRAGE to characterize the AGEs/sRAGE axis strengthened the associations observed. High glycation/sRAGE ratios were associated with 1.3 to 14-fold likelihood of lower AST/ALT ratios. The sum of AGEs/sRAGE ratios accurately distinguished between healthy controls and NAFLD patients (area under the curve of 0.85). Elevated AGEs/sRAGE (>7.8 mmol/pmol) was associated with a 12-fold likelihood of the presence of NAFLD. These findings strengthen the involvement of AGEs-RAGE axis in liver injury and the pathogenesis of NAFLD. Copyright © 2018 Elsevier Inc. All rights reserved.

Correlative fluorescence and scanning transmission electron microscopy of quantum dot-labeled proteins on whole cells in liquid.

PubMed

Peckys, Diana B; Bandmann, Vera; de Jonge, Niels

2014-01-01

Correlative fluorescence microscopy combined with scanning transmission electron microscopy (STEM) of cells fully immersed in liquid is a new methodology with many application areas. Proteins, in live cells immobilized on microchips, are labeled with fluorescent quantum dot nanoparticles. In this protocol, the epidermal growth factor receptor (EGFR) is labeled. The cells are fixed after a selected labeling time, for example, 5 min as needed to form EGFR dimers. The microchip with cells is then imaged with fluorescence microscopy. Thereafter, STEM can be accomplished in two ways. The microchip with the labeled cells and one microchip with a spacer are assembled into a special microfluidic device and imaged with dedicated high-voltage STEM. Alternatively, thin edges of cells can be studied with environmental scanning electron microscopy with a STEM detector, by placing a microchip with cells in a cooled wet environment. © 2014 Elsevier Inc. All rights reserved.

Laboratory studies of in vivo fluorescence of phytoplankton

NASA Technical Reports Server (NTRS)

Brown, C. A., Jr.; Farmer, F. H.; Jarrett, O., Jr.; Staton, W. L.

1978-01-01

A lidar system is developed that uses four selected excitation wavelengths to induce chlorophyll 'a' fluorescence which is indicative of both the concentration and diversity of phytoplankton. The operating principles of the system and the results of measurements of phytoplankton fluorescence in a controlled laboratory environment are presented. A comparative study of results from lidar fluorosensor laboratory tank tests using representative species of phytoplankton in single and multispecies cultures from each of four color groups reveals that (1) there is good correlation between the fluorescence of chlorophyll 'a' remotely simulated and detected by the lidar system and in-situ measurements using four similar excitation wavelengths in a flow-through fluorometer; (2) good correlation exists between the total chlorophyll 'a' calculated from lidar-fluorosensor data and measurements obtained by the Strickland-Parsons method; and (3) the lidar fluorosensor can provide an index of population diversity.

Tracking Silent Hypersensitivity Reactions to Asparaginase during Leukemia Therapy Using Single-Chip Indirect Plasmonic and Fluorescence Immunosensing.

PubMed

Charbonneau, David M; Breault-Turcot, Julien; Sinnett, Daniel; Krajinovic, Maja; Leclerc, Jean-Marie; Masson, Jean-François; Pelletier, Joelle N

2017-12-22

Microbial asparaginase is an essential component of chemotherapy for the treatment of childhood acute lymphoblastic leukemia (cALL). Silent hypersensitivity reactions to this microbial enzyme need to be monitored accurately during treatment to avoid adverse effects of the drug and its silent inactivation. Here, we present a dual-response anti-asparaginase sensor that combines indirect SPR and fluorescence on a single chip to perform ELISA-type immunosensing, and correlate measurements with classical ELISA. Analysis of serum samples from children undergoing cALL therapy revealed a clear correlation between single-chip indirect SPR/fluorescence immunosensing and ELISA used in clinical settings (R 2 > 0.9). We also report that the portable SPR/fluorescence system had a better sensitivity than classical ELISA to detect antibodies in clinical samples with low antigenicity. This work demonstrates the reliability of dual sensing for monitoring clinically relevant antibody titers in clinical serum samples.

Fluorescence spectroscopy for assessment of liver transplantation grafts concerning graft viability and patient survival

NASA Astrophysics Data System (ADS)

Vollet Filho, José D.; da Silveira, Marina R.; Castro-e-Silva, Orlando; Bagnato, Vanderlei S.; Kurachi, Cristina

2015-06-01

Evaluating transplantation grafts at harvest is essential for its success. Laser-induced fluorescence spectroscopy (LIFS) can help monitoring changes in metabolic/structural conditions of tissue during transplantation. The aim of the present study is to correlate LIFSobtained spectra of human hepatic grafts during liver transplantation with post-operative patients' mortality rate and biochemical parameters, establishing a method to exclude nonviable grafts before implantation. Orthotopic liver transplantation, piggyback technique was performed in 15 patients. LIFS was performed under 408nm excitation. Collection was performed immediately after opening donor's abdominal cavity, after cold perfusion, end of back-table period, and 5 min and 1 h after warm perfusion at recipient. Fluorescence information was compared to lactate, creatinine, bilirubin and INR levels and to survival status. LIFS was sensitive to liver changes during transplantation stages. Study-in-progress; initial results indicate correlation between fluorescence and life/death status of patients.

Image correlation microscopy for uniform illumination.

PubMed

Gaborski, T R; Sealander, M N; Ehrenberg, M; Waugh, R E; McGrath, J L

2010-01-01

Image cross-correlation microscopy is a technique that quantifies the motion of fluorescent features in an image by measuring the temporal autocorrelation function decay in a time-lapse image sequence. Image cross-correlation microscopy has traditionally employed laser-scanning microscopes because the technique emerged as an extension of laser-based fluorescence correlation spectroscopy. In this work, we show that image correlation can also be used to measure fluorescence dynamics in uniform illumination or wide-field imaging systems and we call our new approach uniform illumination image correlation microscopy. Wide-field microscopy is not only a simpler, less expensive imaging modality, but it offers the capability of greater temporal resolution over laser-scanning systems. In traditional laser-scanning image cross-correlation microscopy, lateral mobility is calculated from the temporal de-correlation of an image, where the characteristic length is the illuminating laser beam width. In wide-field microscopy, the diffusion length is defined by the feature size using the spatial autocorrelation function. Correlation function decay in time occurs as an object diffuses from its original position. We show that theoretical and simulated comparisons between Gaussian and uniform features indicate the temporal autocorrelation function depends strongly on particle size and not particle shape. In this report, we establish the relationships between the spatial autocorrelation function feature size, temporal autocorrelation function characteristic time and the diffusion coefficient for uniform illumination image correlation microscopy using analytical, Monte Carlo and experimental validation with particle tracking algorithms. Additionally, we demonstrate uniform illumination image correlation microscopy analysis of adhesion molecule domain aggregation and diffusion on the surface of human neutrophils.

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

Measuring and imaging diffusion with multiple scan speed image correlation spectroscopy.

PubMed

Gröner, Nadine; Capoulade, Jérémie; Cremer, Christoph; Wachsmuth, Malte

2010-09-27

The intracellular mobility of biomolecules is determined by transport and diffusion as well as molecular interactions and is crucial for many processes in living cells. Methods of fluorescence microscopy like confocal laser scanning microscopy (CLSM) can be used to characterize the intracellular distribution of fluorescently labeled biomolecules. Fluorescence correlation spectroscopy (FCS) is used to describe diffusion, transport and photo-physical processes quantitatively. As an alternative to FCS, spatially resolved measurements of mobilities can be implemented using a CLSM by utilizing the spatio-temporal information inscribed into the image by the scan process, referred to as raster image correlation spectroscopy (RICS). Here we present and discuss an extended approach, multiple scan speed image correlation spectroscopy (msICS), which benefits from the advantages of RICS, i.e. the use of widely available instrumentation and the extraction of spatially resolved mobility information, without the need of a priori knowledge of diffusion properties. In addition, msICS covers a broad dynamic range, generates correlation data comparable to FCS measurements, and allows to derive two-dimensional maps of diffusion coefficients. We show the applicability of msICS to fluorophores in solution and to free EGFP in living cells.

Correlative cellular ptychography with functionalized nanoparticles at the Fe L-edge

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

Gallagher-Jones, Marcus; Dias, Carlos Sato Baraldi; Pryor, Alan

Precise localization of nanoparticles within a cell is crucial to the understanding of cell-particle interactions and has broad applications in nanomedicine. Here in this paper, we report a proof-of-principle experiment for imaging individual functionalized nanoparticles within a mammalian cell by correlative microscopy. Using a chemically-fixed HeLa cell labeled with fluorescent core-shell nanoparticles as a model system, we implemented a graphene-oxide layer as a substrate to significantly reduce background scattering. We identified cellular features of interest by fluorescence microscopy, followed by scanning transmission X-ray tomography to localize the particles in 3D, and ptychographic coherent diffractive imaging of the fine features inmore » the region at high resolution. By tuning the X-ray energy to the Fe L-edge, we demonstrated sensitive detection of nanoparticles composed of a 22 nm magnetic Fe 3O 4 core encased by a 25-nm-thick fluorescent silica (SiO 2) shell. These fluorescent core-shell nanoparticles act as landmarks and offer clarity in a cellular context. Our correlative microscopy results confirmed a subset of particles to be fully internalized, and high-contrast ptychographic images showed two oxidation states of individual nanoparticles with a resolution of ~16.5 nm. The ability to precisely localize individual fluorescent nanoparticles within mammalian cells will expand our understanding of the structure/function relationships for functionalized nanoparticles.« less

Correlative cellular ptychography with functionalized nanoparticles at the Fe L-edge

DOE PAGES

Gallagher-Jones, Marcus; Dias, Carlos Sato Baraldi; Pryor, Alan; ...

2017-07-06

Precise localization of nanoparticles within a cell is crucial to the understanding of cell-particle interactions and has broad applications in nanomedicine. Here in this paper, we report a proof-of-principle experiment for imaging individual functionalized nanoparticles within a mammalian cell by correlative microscopy. Using a chemically-fixed HeLa cell labeled with fluorescent core-shell nanoparticles as a model system, we implemented a graphene-oxide layer as a substrate to significantly reduce background scattering. We identified cellular features of interest by fluorescence microscopy, followed by scanning transmission X-ray tomography to localize the particles in 3D, and ptychographic coherent diffractive imaging of the fine features inmore » the region at high resolution. By tuning the X-ray energy to the Fe L-edge, we demonstrated sensitive detection of nanoparticles composed of a 22 nm magnetic Fe 3O 4 core encased by a 25-nm-thick fluorescent silica (SiO 2) shell. These fluorescent core-shell nanoparticles act as landmarks and offer clarity in a cellular context. Our correlative microscopy results confirmed a subset of particles to be fully internalized, and high-contrast ptychographic images showed two oxidation states of individual nanoparticles with a resolution of ~16.5 nm. The ability to precisely localize individual fluorescent nanoparticles within mammalian cells will expand our understanding of the structure/function relationships for functionalized nanoparticles.« less

Fluorescence lifetime intravascular ultrasound (FLIm-IVUS) and the quest to discriminate between early and advanced lipid cores in atherosclerosis (Conference Presentation)

NASA Astrophysics Data System (ADS)

Phipps, Jennifer E.; Bec, Julien; Vela, Deborah; Buja, L. Maximilian; Southard, Jeffrey A.; Margulies, Kenneth B.; Marcu, Laura

2017-02-01

FL-IVUS combines intravascular ultrasound with fluorescence lifetime imaging to obtain morphologic and biochemical details from the arterial wall. Ultrasound measurements alone provide morphologic information (plaque burden, remodeling index and presence of calcium). Fluorescence lifetime can determine the presence of a thick fibrous cap, macrophage infiltration, and lipid cores beneath thin fibrous caps. These details are important to assess plaque vulnerability. In this study, we focused on the ability of FL-IVUS to differentiate between early and advanced lipid cores-advanced cores are vulnerable to rupture. We imaged N=12 ex vivo human coronary arteries and performed hematoxylin and eosin, Movat's pentachrome and CD68 immunohistochemistry at 500 micron intervals throughout the length of the vessels. We found only N=1 thin-capped fibroatheroma (TCFA) with an advanced necrotic core and N=7 cases of foam cell infiltration, early lipid cores or deep necrotic cores. IVUS was able to observe the increased plaque burden and calcification of the advanced and deep necrotic cores, but could not identify early lipid cores, foam cell infiltration or discriminate between deep necrotic cores and TCFA. The addition of FLIm to IVUS allowed the TCFA to be discriminated from early lipid accumulation, particularly at 542+/-50 nm (355 nm pulsed excitation): 7.6 +/- 0.5 ns compared to 6.6 +/- 0.4 ns, respectively (P<0.001 by ANOVA analysis). These differences need to be validated in a larger cohort, but exist due to specific lipid content in the necrotic core as well as increased extracellular matrix in early lesions.

Development of fluorescent probes based on protection-deprotection of the key functional groups for biological imaging.

PubMed

Tang, Yonghe; Lee, Dayoung; Wang, Jiaoliang; Li, Guanhan; Yu, Jinghua; Lin, Weiying; Yoon, Juyoung

2015-08-07

Recently, the strategy of protection-deprotection of functional groups has been widely employed to design fluorescent probes, as the protection-deprotection of functional groups often induces a marked change in electronic properties. Significant advances have been made in the development of analyte-responsive fluorescent probes based on the protection-deprotection strategy. In this tutorial review, we highlight the representative examples of small-molecule based fluorescent probes for bioimaging, which are operated via the protection-deprotection of key functional groups such as aldehyde, hydroxyl, and amino functional groups reported from 2010 to 2014. The discussion includes the general protection-deprotection methods for aldehyde, hydroxyl, or amino groups, as well as the design strategies, sensing mechanisms, and deprotection modes of the representative fluorescent imaging probes applied to bio-imaging.

Emerging biomedical applications of time-resolved fluorescence spectroscopy

NASA Astrophysics Data System (ADS)

Lakowicz, Joseph R.; Szmacinski, Henryk; Koen, Peter A.

1994-07-01

Time-resolved fluorescence spectroscopy is presently regarded as a research tool in biochemistry, biophysics, and chemical physics. Advances in laser technology, the development of long-wavelength probes, and the use of lifetime-based methods are resulting in the rapid migration of time-resolved fluorescence to the clinical chemistry lab, to the patient's bedside, to flow cytometers, to the doctor's office, and even to home health care. Additionally, time-resolved imaging is now a reality in fluorescence microscopy, and will provide chemical imaging of a variety of intracellular analytes and/or cellular phenomena. In this overview paper we attempt to describe some of the opportunities available using chemical sensing based on fluorescence lifetimes, and to predict those applications of lifetime-based sensing which are most likely in the near future.

Chamber catalogues of optical and fluorescent signatures distinguish bioaerosol classes

NASA Astrophysics Data System (ADS)

Hernandez, Mark; Perring, Anne E.; McCabe, Kevin; Kok, Greg; Granger, Gary; Baumgardner, Darrel

2016-07-01

Rapid bioaerosol characterization has immediate applications in the military, environmental and public health sectors. Recent technological advances have facilitated single-particle detection of fluorescent aerosol in near real time; this leverages controlled ultraviolet exposures with single or multiple wavelengths, followed by the characterization of associated fluorescence. This type of ultraviolet induced fluorescence has been used to detect airborne microorganisms and their fragments in laboratory studies, and it has been extended to field studies that implicate bioaerosol to compose a substantial fraction of supermicron atmospheric particles. To enhance the information yield that new-generation fluorescence instruments can provide, we report the compilation of a referential aerobiological catalogue including more than 50 pure cultures of common airborne bacteria, fungi and pollens, recovered at water activity equilibrium in a mesoscale chamber (1 m3). This catalogue juxtaposes intrinsic optical properties and select bandwidths of fluorescence emissions, which manifest to clearly distinguish between major classes of airborne microbes and pollens.

Quantitative analysis of tear film fluorescence and discomfort during tear film instability and thinning.

PubMed

Begley, Carolyn; Simpson, Trefford; Liu, Haixia; Salvo, Eliza; Wu, Ziwei; Bradley, Arthur; Situ, Ping

2013-04-12

The purpose of this study was to test the association between tear film fluorescence changes during tear break-up (TBU) or thinning and the concurrent ocular sensory response. Sixteen subjects kept one eye open as long as possible (MBI), indicated their discomfort level continuously, and rated ocular sensations of irritation, stinging, burning, pricking, and cooling using visual analog scales (VAS). Fluorescence of the tear film was quantified by a pixel-based analysis of the median pixel intensity (PI), TBU, and percentage of dark pixels (DarkPix) over time. A cutoff of 5% TBU was used to divide subjects into either break-up (BU) or minimal break-up (BUmin) groups. Tear film fluorescence decreased (median PI) and the percentage of TBU and DarkPix increased in all trials, with the rate significantly greater in the BU than the BUmin group (Mann-Whitney U test, P < 0.05). The rate of increasing discomfort during trials was highly correlated with the rate of decrease in median PI and developing TBU (Spearman's, r ≥ 0.70). Significant correlations were found between corneal fluorescence, MBI, and sensory measures. Concentration quenching of fluorescein dye with tear film thinning best explains decreasing tear film fluorescence during trials. This was highly correlated with increasing ocular discomfort, suggesting that both tear film thinning and TBU stimulate underlying corneal nerves, although TBU produced more rapid stimulation. Slow increases in tear film hyperosmolarity may cause the gradual increase in discomfort during slow tear film thinning, whereas the sharp increases in discomfort during TBU suggest a more complex stimulus.

Fluorescence fingerprinting properties for exploring water-soluble organic compounds in PM2.5 in an industrial city of northwest China

NASA Astrophysics Data System (ADS)

Qin, Juanjuan; Zhang, Leiming; Zhou, Xueming; Duan, Jingchun; Mu, Situ; Xiao, Kang; Hu, Jingnan; Tan, Jihua

2018-07-01

Excitation-emission matrix (EEM) spectra were used to analyze the fluorescence properties of water-soluble organic compounds (WSOC) in PM2.5 during winter and summer seasons in Lanzhou city, northwest China. Protein-like substances, humic-like substances and microbial by-products were found to be the main fluorophoric organic matter. Humification index (HIX), biological index (BIX) and fluorescence index (FI) were 1.2 ± 0.1, 1.4 ± 0.1 and 1.7 ± 0.2 in winter and 2.0 ± 0.3, 1.3 ± 0.1 and 1.2 ± 0.4 in summer, respectively, indicating higher aromaticity of WSOC in summer and terrestrial biogenic sources of WSOC in both seasons. Strong correlations were found between regional average fluorescence intensities (RAFI) at the fluorescence regions of II-V and water-soluble inorganic ions (K+, Cl-, NO3- and SO42-) in winter with the Spearman correlation coefficients ρ being larger than 0.7 and mostly around 0.9, suggesting significant contributions of bioaerosols, coal combustion and vehicle exhaust to PM2.5. Moderate correlations were found between RAFI and K+, Cl-, and NO3- in summer, indicating the more important roles of biogenic and vehicle exhaust than coal combustion sources in summer. High temperature in winter was conducive to and in summer prohibitive to polycondensation of WSOC, indicating the existence of an optimum temperature for such a process to occur. High relative humidity was unfavorable to the formation of fluorescent WSOC.

Polarization effects in cutaneous autofluorescent spectra

NASA Astrophysics Data System (ADS)

Borisova, E.; Angelova, L.; Jeliazkova, Al.; Genova, Ts.; Pavlova, E.; Troyanova, P.; Avramov, L.

2014-05-01

Used polarized light for fluorescence excitation one could obtain response related to the anisotropy features of extracellular matrix. The fluorophore anisotropy is attenuated during lesions' growth and level of such decrease could be correlated with the stage of tumor development. Our preliminary investigations are based on in vivo point-by-point measurements of excitation-emission matrices (EEM) from healthy volunteers skin on different ages and from different anatomical places using linear polarizer and analyzer for excitation and emission light detected. Measurements were made using spectrofluorimeter FluoroLog 3 (HORIBA Jobin Yvon, France) with fiber-optic probe in steady-state regime using excitation in the region of 280-440 nm. Three different situations were evaluated and corresponding excitation-emission matrices were developed - with parallel and perpendicular positions for linear polarizer and analyzer, and without polarization of excitation and fluorescence light detected from a forearm skin surface. The fluorescence spectra obtained reveal differences in spectral intensity, related to general attenuation, due to filtering effects of used polarizer/analyzer couple. Significant spectral shape changes were observed for the complex autofluorescence signal detected, which correlated with collagen and protein cross-links fluorescence, that could be addressed to the tissue extracellular matrix and general condition of the skin investigated, due to morphological destruction during lesions' growth. A correlation between volunteers' age and the fluorescence spectra detected was observed during our measurements. Our next step is to increase developed initial database and to evaluate all sources of intrinsic fluorescent polarization effects and found if they are significantly altered from normal skin to cancerous state of the tissue, this way to develop a non-invasive diagnostic tool for dermatological practice.

Probing GFP-actin diffusion in living cells using fluorescence correlation spectroscopy.

PubMed

Engelke, Hanna; Heinrich, Doris; Rädler, Joachim O

2010-12-22

The cytoskeleton of eukaryotic cells is continuously remodeled by polymerization and depolymerization of actin. Consequently, the relative content of polymerized filamentous actin (F-actin) and monomeric globular actin (G-actin) is subject to temporal and spatial fluctuations. Since fluorescence correlation spectroscopy (FCS) can measure the diffusion of fluorescently labeled actin it seems likely that FCS allows us to determine the dynamics and hence indirectly the structural properties of the cytoskeleton components with high spatial resolution. To this end we investigate the FCS signal of GFP-actin in living Dictyostelium discoideum cells and explore the inherent spatial and temporal signatures of the actin cytoskeleton. Using the free green fluorescent protein (GFP) as a reference, we find that actin diffusion inside cells is dominated by G-actin and slower than diffusion in diluted cell extract. The FCS signal in the dense cortical F-actin network near the cell membrane is probed using the cytoskeleton protein LIM and is found to be slower than cytosolic G-actin diffusion. Furthermore, we show that polymerization of the cytoskeleton induced by Jasplakinolide leads to a substantial decrease of G-actin diffusion. Pronounced fluctuations in the distribution of the FCS correlation curves can be induced by latrunculin, which is known to induce actin waves. Our work suggests that the FCS signal of GFP-actin in combination with scanning or spatial correlation techniques yield valuable information about the local dynamics and concomitant cytoskeletal properties.

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.

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.

Assessment of β-zone peripapillary atrophy by optical coherence tomography and scanning laser ophthalmoscopy imaging in glaucoma patients

PubMed Central

Seidensticker, Florian; Reznicek, Lukas; Mann, Thomas; Hübert, Irene; Kampik, Anselm; Ulbig, Michael; Hirneiss, Christoph; Neubauer, Aljoscha S; Kernt, Marcus

2014-01-01

Purpose To assess β-zone peripapillary atrophy (β-PPA) using spectral domain optical coherence tomography (SD-OCT), scanning laser ophthalmoscopy (SLO), and fundus auto-fluorescence (FAF) imaging in patients with primary open-angle glaucoma with advanced glaucomatous visual field defects. Methods A consecutive, prospective series of 82 study eyes with primary open-angle glaucoma were included in this study. All study participants underwent a full ophthalmic examination followed by SD-OCT, wide-field SLO, and FAF imaging of the optic nerve head and the peripapillary region. Results Eighty-four glaucomatous eyes were included in our prospective study. Correlation analyses for horizontally and vertically obtained β-PPA for all three imaging modalities (color SLO, FAF, and SD-OCT) revealed highest correlations between FAF and color SLO (Pearson correlation coefficient: 0.904 [P<0.001] for horizontal β-PPA and 0.786 [P<0.001] for vertical β-PPA). Bland–Altman plotting revealed highest agreements between color SLO and FAF, with −2.1 pixels ±1.96 standard deviation (SD) for horizontal β-PPA, SD: 10.5 pixels and 2.4 pixels ±1.96 SD for vertical β-PPA. Conclusion β-PPA can be assessed using en-face SLO and cross-sectional SD-OCT imaging. Correlation analyses revealed highest correlations between color SLO and FAF imaging, while correlations between SLO and SD-OCT were weak. A more precise structural definition of β-PPA is needed. PMID:25061270

Use of COD, TOC, and Fluorescence Spectroscopy to Estimate BOD in Wastewater.

PubMed

Christian, Evelyn; Batista, Jacimaria R; Gerrity, Daniel

2017-02-01

  Common to all National Pollutant Discharge Elimination System (NPDES) permits in the United States is a limit on biochemical oxygen demand (BOD). Chemical oxygen demand (COD), total organic carbon (TOC), and fluorescence spectroscopy are also capable of quantifying organic content, although the mechanisms of quantification and the organic fractions targeted differ for each test. This study explores correlations between BOD5 and these alternate test procedures using facility influent, primary effluent, and facility effluent samples from a full-scale water resource recovery facility. Relative reductions of the water quality parameters proved to be strong indicators of their suitability as surrogates for BOD5. Suitable correlations were generally limited to the combined datasets for the three sampling locations or the facility effluent alone. COD exhibited relatively strong linear correlations with BOD5 when considering the three sample points (r = 0.985) and the facility effluent alone (r = 0.914), while TOC exhibited a suitable linear correlation with BOD5 in the facility effluent (r = 0.902). Exponential regressions proved to be useful for estimating BOD5 based on TOC or fluorescence (r > 0.95).

Fluorescence correlation spectroscopy and fluorescence cross-correlation spectroscopy reveal the cytoplasmic origination of loaded nuclear RISC in vivo in human cells

PubMed Central

Mütze, Jörg; Staroske, Wolfgang; Weinmann, Lasse; Höck, Julia; Crell, Karin; Meister, Gunter; Schwille, Petra

2008-01-01

Studies of RNA interference (RNAi) provide evidence that in addition to the well-characterized cytoplasmic mechanisms, nuclear mechanisms also exist. The mechanism by which the nuclear RNA-induced silencing complex (RISC) is formed in mammalian cells, as well as the relationship between the RNA silencing pathways in nuclear and cytoplasmic compartments is still unknown. Here we show by applying fluorescence correlation and cross-correlation spectroscopy (FCS/FCCS) in vivo that two distinct RISC exist: a large ∼3 MDa complex in the cytoplasm and a 20-fold smaller complex of ∼158 kDa in the nucleus. We further show that nuclear RISC, consisting only of Ago2 and a short RNA, is loaded in the cytoplasm and imported into the nucleus. The loaded RISC accumulates in the nucleus depending on the presence of a target, based on an miRNA-like interaction with impaired cleavage of the cognate RNA. Together, these results suggest a new RISC shuttling mechanism between nucleus and cytoplasm ensuring concomitant gene regulation by small RNAs in both compartments. PMID:18842624

Fluorescence correlation spectroscopy and fluorescence cross-correlation spectroscopy reveal the cytoplasmic origination of loaded nuclear RISC in vivo in human cells.

PubMed

Ohrt, Thomas; Mütze, Jörg; Staroske, Wolfgang; Weinmann, Lasse; Höck, Julia; Crell, Karin; Meister, Gunter; Schwille, Petra

2008-11-01

Studies of RNA interference (RNAi) provide evidence that in addition to the well-characterized cytoplasmic mechanisms, nuclear mechanisms also exist. The mechanism by which the nuclear RNA-induced silencing complex (RISC) is formed in mammalian cells, as well as the relationship between the RNA silencing pathways in nuclear and cytoplasmic compartments is still unknown. Here we show by applying fluorescence correlation and cross-correlation spectroscopy (FCS/FCCS) in vivo that two distinct RISC exist: a large approximately 3 MDa complex in the cytoplasm and a 20-fold smaller complex of approximately 158 kDa in the nucleus. We further show that nuclear RISC, consisting only of Ago2 and a short RNA, is loaded in the cytoplasm and imported into the nucleus. The loaded RISC accumulates in the nucleus depending on the presence of a target, based on an miRNA-like interaction with impaired cleavage of the cognate RNA. Together, these results suggest a new RISC shuttling mechanism between nucleus and cytoplasm ensuring concomitant gene regulation by small RNAs in both compartments.

Fluorescence correlation spectroscopy: principles and applications.

PubMed

Bacia, Kirsten; Haustein, Elke; Schwille, Petra

2014-07-01

Fluorescence correlation spectroscopy (FCS) is used to study the movements and the interactions of biomolecules at extremely dilute concentrations, yielding results with good spatial and temporal resolutions. Using a number of technical developments, FCS has become a versatile technique that can be used to study a variety of sample types and can be advantageously combined with other methods. Unlike other fluorescence-based techniques, the analysis of FCS data is not based on the average intensity of the fluorescence emission but examines the minute intensity fluctuations caused by spontaneous deviations from the mean at thermal equilibrium. These fluctuations can result from variations in local concentrations owing to molecular mobility or from characteristic intermolecular or intramolecular reactions of fluorescently labeled biomolecules present at low concentrations. Here, we provide a basic introduction to FCS, including its technical development and theoretical basis, experimental setup of an FCS system, adjustment of a setup, data acquisition, and analysis of FCS measurements. Finally, the application of FCS to the study of lipid bilayer membranes and to living cells is discussed. © 2014 Cold Spring Harbor Laboratory Press.

Determination of adenine based on the fluorescence recovery of the L-Tryptophan-Cu(2+) complex.

PubMed

Duan, Ruilin; Li, Chunyan; Liu, Shaopu; Liu, Zhongfang; Li, Yuanfang; Yuan, Yusheng; Hu, Xiaoli

2016-01-05

A simple and sensitive method for determination of adenine was developed based on fluorescence quenching and recovery of L-Tryptophan (L-Trp). The fluorescence of L-Trp could efficiently quenched by copper ion compared with other common metal ions. Upon addition of adenine (Ade) in L-Trp-Cu(II) system, the fluorescence was reoccurred. Under the optimum conditions, the recovery fluorescence intensity was linearly correlated with the concentration of adenine in the range from 0.34 to 25.0μmolL(-1), with a correlation coefficient (R(2)) of 0.9994. The detection limit (3σ/k) was 0.046μmolL(-1), indicating that this method could applied to detect trace adenine. In this study, amino acids including L-Trp, D-Trp, L-Tyr, D-Tyr, L-Phe, D-Phe were investigated and only L-Trp could well chelated copper ion. Additionally, the mechanism of quench and recovery also were discussed and the method was successfully applied to detect the adenine in DNA with satisfactory results. Copyright © 2015 Elsevier B.V. All rights reserved.

Superiorities of time-correlated single-photon counting against standard fluorimetry in exploiting the potential of fluorochromized oligonucleotide probes for biomedical investigation

NASA Astrophysics Data System (ADS)

Lamperti, Marco; Nardo, Luca; Bondani, Maria

2015-05-01

Site-specific fluorescence-resonance-energy-transfer donor-acceptor dual-labelled oligonucleotide probes are widely used in state-of-art biotechnological applications. Such applications include their usage as primers in polymerase chain reaction. However, the steady-state fluorescence intensity signal emitted by these molecular tools strongly depends from the specificities of the probe conformation. For this reason, the information which can be reliably inferred by steady-state fluorimetry performed on such samples is forcedly confined to a semi-qualitative level. Namely, fluorescent emission is frequently used as ON/OFF indicator of the probe hybridization state, i.e. detection of fluorescence signals indicates either hybridization to or detachment from the template DNA of the probe. Nonetheless, a fully quantitative analysis of their fluorescence emission properties would disclose other exciting applications of dual-labelled probes in biosensing. Here we show how time-correlated single-photon counting can be applied to get rid of the technical limitations and interpretational ambiguities plaguing the intensity analysis, and to derive information on the template DNA reaching single-base.

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.

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

Shcheslavskiy, V. I.; Institute of Biomedical Technologies, Nizhny Novgorod State Medical Academy, Minin and Pozharsky Square, 10/1, Nizhny Novgorod 603005; Neubauer, A.

We present a lifetime imaging technique that simultaneously records the fluorescence and phosphorescence lifetime images in confocal laser scanning systems. It is based on modulating a high-frequency pulsed laser synchronously with the pixel clock of the scanner, and recording the fluorescence and phosphorescence signals by multidimensional time-correlated single photon counting board. We demonstrate our technique on the recording of the fluorescence/phosphorescence lifetime images of human embryonic kidney cells at different environmental conditions.

Chemistry Is Dead. Long Live Chemistry!

PubMed

Lavis, Luke D

2017-10-03

Chemistry, once king of fluorescence microscopy, was usurped by the field of fluorescent proteins. The increased demands of modern microscopy techniques on the "photon budget" require better and brighter fluorophores, causing a renewed interest in synthetic dyes. Here, we review the recent advances in biochemistry, protein engineering, and organic synthesis that have allowed a triumphant return of chemical fluorophores to modern biological imaging.

Synchrotron-based X-ray Fluorescence Microscopy in Conjunction with Nanoindentation to Study Molecular-Scale Interactions of Phenol–Formaldehyde in Wood Cell Walls

Treesearch

Joseph E. Jakes; Christopher G. Hunt; Daniel J. Yelle; Linda Lorenz; Kolby Hirth; Sophie-Charlotte Gleber; Stefan Vogt; Warren Grigsby; Charles R. Frihart

2015-01-01

Understanding and controlling molecular-scale interactions between adhesives and wood polymers are critical to accelerate the development of improved adhesives for advanced wood-based materials. The submicrometer resolution of synchrotron-based X-ray fluorescence microscopy (XFM) was found capable of mapping and quantifying infiltration of Br-labeled phenol−...

Fluorescence Spectroscopy of tRNA[superscript Phe] Y Base in the Presence of Mg[superscript 2+] and Small Molecule Ligands

ERIC Educational Resources Information Center

Kirk, Sarah R.; Silverstein, Todd P.; McFarlane Holman, Karen L.

2008-01-01

This laboratory project is one component of a semester-long advanced biochemistry laboratory course that uses several complementary techniques to study tRNA[superscript Phe] conformational changes induced by ligand binding. In this article we describe a set of experiments in which students use fluorescence spectroscopy to study tRNA[superscript…

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.

The effect of trypan blue treatment on autofluorescence of fixed cells.

PubMed

Shilova, Olga N; Shilov, Evgeny S; Deyev, Sergey M

2017-09-01

Controlling background fluorescence remains an important challenge in flow cytometry, as autofluorescence can interfere with the detection of chromophores. Furthermore, experimental procedures can also affect cellular fluorescence in certain regions of the emission spectrum. In this work, the effects of fixation, permeabilization, and heating on cellular autofluorescence are analyzed in various spectral regions, along with the influence of trypan blue as a quenching dye for these treatments. The impact of these procedures on the staining of SK-BR-3 cells with a dim green fluorophore, a miniSOG (mini Singlet Oxygen Generator) flavoprotein in the form of the recombinant protein DARPin-miniSOG, is also evaluated. The data presented here indicate that fixation of certain types of cells leads to noticeable increase of the autofluorescence. Our results also suggest that trypan blue should be used as an autofluorescence quencher only with bright green emitters since it interferes with the fluorescent signal in a longer-wavelength region of the spectrum and as a result causes reduction of the signal from dim green fluorescent agents. © 2017 International Society for Advancement of Cytometry. © 2017 International Society for Advancement of Cytometry.

The endogenous fluorescence of fibroblast in collagen gels as indicator of stiffness of the extracellular matrix

NASA Astrophysics Data System (ADS)

Padilla-Martinez, J. P.; Ortega-Martinez, A.; Franco, W.

2016-03-01

The stiffness or rigidity of the extracellular matrix (ECM) regulates cell response. Established mechanical tests to measure stiffness, such as indentation and tensile tests, are invasive and destructive to the sample. Endogenous or native molecules to cells and ECM components, like tryptophan and cross-links of collagen, display fluorescence upon irradiation with ultraviolet light. Most likely, the concentration of these endogenous fluorophores changes as the stiffness of the ECM changes. In this work we investigate the endogenous fluorescence of collagen gels containing fibroblasts as a non-invasive non-destructive method to measure stiffness of the ECM. Human fibroblast cells were cultured in three-dimensional gels of type I collagen (50,000 cells/ml). This construct is a simple model of tissue contraction. During contraction, changes in the excitation-emission matrix (a fluorescence map in the 240-520/290-530 nm range) of constructs were measured with a spectrofluoremeter, and changes in stiffness were measured with a standard indentation test over 16 days. Results show that a progressive increase in fluorescence of the 290/340 nm excitation-emission pair correlates with a progressive increase in stiffness (r=0.9, α=0.5). The fluorescence of this excitation-emission pair is ascribed to tryptophan and variations in the fluorescence of this pair correlate with cellular proliferation. In this tissue model, the endogenous functional fluorescence of proliferating fibroblast cells is a biomechanical marker of stiffness of the ECM.

Relating Trp-Glu dipeptide fluorescence to molecular conformation: the role of the discrete Chi 1 and Chi 2 angles.

PubMed

Eisenberg, Azaria Solomon; Juszczak, Laura J

2013-07-05

Molecular dynamics (MD), coupled with fluorescence data for charged dipeptides of tryptophanyl glutamic acid (Trp-Glu), reveal a detailed picture of how specific conformation affects fluorescence. Fluorescence emission spectra and time-resolved emission measurements have been collected for all four charged species. MD simulations 20 to 30 ns in length have also been carried out for the Trp-Glu species, as simulation provides aqueous phase conformational data that can be correlated with the fluorescence data. The calculations show that each dipeptide species is characterized by a similar set of six, discrete Chi 1, Chi 2 dihedral angle pairs. The preferred Chi 1 angles--60°, 180°, and 300°--play the significant role in positioning the terminal amine relative to the indole ring. A Chi 1 angle of 60° results in the arching of the backbone over the indole ring and no interaction of the ring with the terminal amine. Chi 1 values of 180° and 300° result in an extension of the backbone away from the indole ring and a NH3 cation-π interaction with indole. This interaction is believed responsible for charge transfer quenching. Two fluorescence lifetimes and their corresponding amplitudes correlate with the Chi 1 angle probability distribution for all four charged Trp-Glu dipeptides. Fluorescence emission band maxima are also consistent with the proposed pattern of terminal amine cation quenching of fluorescence. Copyright © 2013 Wiley Periodicals, Inc.

Revealing the Effects of Nanoscale Membrane Curvature on Lipid Mobility.

PubMed

Kabbani, Abir Maarouf; Woodward, Xinxin; Kelly, Christopher V

2017-10-18

Recent advances in nanoengineering and super-resolution microscopy have enabled new capabilities for creating and observing membrane curvature. However, the effects of curvature on single-lipid diffusion have yet to be revealed. The simulations presented here describe the capabilities of varying experimental methods for revealing the effects of nanoscale curvature on single-molecule mobility. Traditionally, lipid mobility is revealed through fluorescence recovery after photobleaching (FRAP), fluorescence correlation spectroscopy (FCS), and single particle tracking (SPT). However, these techniques vary greatly in their ability to detect the effects of nanoscale curvature on lipid behavior. Traditionally, FRAP and FCS depend on diffraction-limited illumination and detection. A simulation of FRAP shows minimal effects on lipids diffusion due to a 50 nm radius membrane bud. Throughout the stages of the budding process, FRAP detected minimal changes in lipid recovery time due to the curvature versus flat membrane. Simulated FCS demonstrated small effects due to a 50 nm radius membrane bud that was more apparent with curvature-dependent lipid mobility changes. However, SPT achieves a sub-diffraction-limited resolution of membrane budding and lipid mobility through the identification of the single-lipid positions with ≤15 nm spatial and ≤20 ms temporal resolution. By mapping the single-lipid step lengths to locations on the membrane, the effects of membrane topography and curvature could be correlated to the effective membrane viscosity. Single-fluorophore localization techniques, such SPT, can detect membrane curvature and its effects on lipid behavior. These simulations and discussion provide a guideline for optimizing the experimental procedures in revealing the effects of curvature on lipid mobility and effective local membrane viscosity.

Fluorescence of fungi in superficial and deep fungal infections

PubMed Central

Elston, Dirk M

2001-01-01

Background Fluorescence of many fungi is noted when H&E stained sections are examined under a fluorescent microscope. In theory, this phenomenon could aid in the diagnosis of cutaneous and disseminated fungal infections without the delay associated with special stains. Seventy-six cases of superficial and deep fungal infections and 3 cases of protothecosis were studied to determine the clinical usefulness of this technique. Results In most cases, fluorescence was noted, but was not intense. Fluorescence of fungi did not correlate with the age of the specimen. In most cases, organisms in H&E stained sections were more easily identified with routine light microscopy than with fluorescent microscopy. Conclusion This report suggests that in H&E stained skin specimens, fluorescent microscopy is of little benefit in the identification of fungal organisms. PMID:11602016

Correlative FRET: new method improves rigor and reproducibility in determining distances within synaptic nanoscale architecture

NASA Astrophysics Data System (ADS)

Shinogle-Decker, Heather; Martinez-Rivera, Noraida; O'Brien, John; Powell, Richard D.; Joshi, Vishwas N.; Connell, Samuel; Rosa-Molinar, Eduardo

2018-02-01

A new correlative Förster Resonance Energy Transfer (FRET) microscopy method using FluoroNanogold™, a fluorescent immunoprobe with a covalently attached Nanogold® particle (1.4nm Au), overcomes resolution limitations in determining distances within synaptic nanoscale architecture. FRET by acceptor photobleaching has long been used as a method to increase fluorescence resolution. The transfer of energy from a donor to an acceptor generally occurs between 10-100Å, which is the relative distance between the donor molecule and the acceptor molecule. For the correlative FRET microscopy method using FluoroNanogold™, we immuno-labeled GFP-tagged-HeLa-expressing Connexin 35 (Cx35) with anti-GFP and with anti-Cx35/36 antibodies, and then photo-bleached the Cx before processing the sample for electron microscopic imaging. Preliminary studies reveal the use of Alexa Fluor® 594 FluoroNanogold™ slightly increases FRET distance to 70Å, in contrast to the 62.5Å using AlexaFluor 594®. Preliminary studies also show that using a FluoroNanogold™ probe inhibits photobleaching. After one photobleaching session, Alexa Fluor 594® fluorescence dropped to 19% of its original fluorescence; in contrast, after one photobleaching session, Alexa Fluor 594® FluoroNanogold™ fluorescence dropped to 53% of its original intensity. This result confirms that Alexa Fluor 594® FluoroNanogold™ is a much better donor probe than is Alexa Fluor 594®. The new method (a) creates a double confirmation method in determining structure and orientation of synaptic architecture, (b) allows development of a two-dimensional in vitro model to be used for precise testing of multiple parameters, and (c) increases throughput. Future work will include development of FluoroNanogold™ probes with different sizes of gold for additional correlative microscopy studies.

INFLUENCE OF ROOT COLONIZING BACTERIA ON THE DEFENSE RESPONSES OF BEAN

EPA Science Inventory

Colonization of plant roots by fluorescent pseudomonads has been correlated with disease suppression. ne mechanism may involve altered defense responses in the plant upon colonization. ltered defense responses were observed in bean (Phaseolus vulgaris) inoculated with fluorescent...

Dynamics of diamond nanoparticles in solution and cells.

PubMed

Neugart, Felix; Zappe, Andrea; Jelezko, Fedor; Tietz, C; Boudou, Jean Paul; Krueger, Anke; Wrachtrup, Jörg

2007-12-01

The fluorescence and motional dynamics of single diamond nanocrystals in buffer solution and in living cells is investigated. Stable hydrosols of nanodiamonds in buffer solutions are investigated by fluorescence correlation spectroscopy. Measurement of the effective hydrodynamic radius yields particles of 48 nm diameter, which is in excellent agreement with atomic force microscopy measurements made on the same particles. Fluorescence correlation spectroscopy measurements indicate that nanocrystals easily form aggregates when the buffer pH is changed. This tendency is reduced when the surface of the diamonds is covered with surfactants. Upon incubation, cells spontaneously take up nanocrystals that uniformly distribute in cells. Most of the particles get immobilized within a few minutes. The binding of streptavidin to biotinylated aggregates of 4 nm diameter nanodiamonds is demonstrated.

Trapping, Deformation, and Rotation of Giant Unilamellar Vesicles in Octode Dielectrophoretic Field Cages

PubMed Central

Korlach, J.; Reichle, C.; Müller, T.; Schnelle, T.; Webb, W. W.

2005-01-01

The behavior of freestanding lipid bilayer membranes under the influence of dielectric force potentials was studied by trapping, holding, and rotating individual giant unilamellar vesicles (GUVs) inside dielectrophoretic microfield cages. Using laser scanning confocal microscopy and three-dimensional image reconstructions of GUVs labeled with fluorescent membrane probes, field strength and frequency-dependent vesicle deformations were observed which are explained by calculations of the dielectric force potentials inside the cage. Dynamical membrane properties under the influence of the field cage were studied by fluorescence correlation spectroscopy, circumventing potential artifacts associated with measurements involving GUV immobilization on support surfaces. Lipid transport could be accelerated markedly by the applied fields, aided by hydrodynamic fluid streaming which was also studied by fluorescence correlation spectroscopy. PMID:15863477

Advances in Light Microscopy for Neuroscience

PubMed Central

Wilt, Brian A.; Burns, Laurie D.; Ho, Eric Tatt Wei; Ghosh, Kunal K.; Mukamel, Eran A.

2010-01-01

Since the work of Golgi and Cajal, light microscopy has remained a key tool for neuroscientists to observe cellular properties. Ongoing advances have enabled new experimental capabilities using light to inspect the nervous system across multiple spatial scales, including ultrastructural scales finer than the optical diffraction limit. Other progress permits functional imaging at faster speeds, at greater depths in brain tissue, and over larger tissue volumes than previously possible. Portable, miniaturized fluorescence microscopes now allow brain imaging in freely behaving mice. Complementary progress on animal preparations has enabled imaging in head-restrained behaving animals, as well as time-lapse microscopy studies in the brains of live subjects. Mouse genetic approaches permit mosaic and inducible fluorescence-labeling strategies, whereas intrinsic contrast mechanisms allow in vivo imaging of animals and humans without use of exogenous markers. This review surveys such advances and highlights emerging capabilities of particular interest to neuroscientists. PMID:19555292

Technological applications arising from the interactions of DNA bases with metal ions.

PubMed

Park, Ki Soo; Park, Hyun Gyu

2014-08-01

An intense interest has grown in the unique interactions of nucleic acids with metal ions, which lead to the formation of metal-base pairs and the generation of fluorescent nanomaterials. In this review, different types of metal-base pairs, especially those formed from naturally occurring nucleosides, are described with emphasis also being given to recent advances made in employing these complexes to govern enzymatic reactions. The review also contains a comprehensive description of DNA-templated inorganic nanomaterials such as silver nanoclusters which possess excellent fluorescence properties. Finally, a summary is given about how these materials have led to recent advances in the field of nanobiotechnology. Copyright © 2013 Elsevier Ltd. All rights reserved.

PyCorrFit-generic data evaluation for fluorescence correlation spectroscopy.

PubMed

Müller, Paul; Schwille, Petra; Weidemann, Thomas

2014-09-01

We present a graphical user interface (PyCorrFit) for the fitting of theoretical model functions to experimental data obtained by fluorescence correlation spectroscopy (FCS). The program supports many data file formats and features a set of tools specialized in FCS data evaluation. The Python source code is freely available for download from the PyCorrFit web page at http://pycorrfit.craban.de. We offer binaries for Ubuntu Linux, Mac OS X and Microsoft Windows. © The Author 2014. Published by Oxford University Press.

Two-Photon Fluorescence Correlation Spectroscopy

NASA Technical Reports Server (NTRS)

Zimmerli, Gregory A.; Fischer, David G.

2002-01-01

We will describe a two-photon microscope currently under development at the NASA Glenn Research Center. It is composed of a Coherent Mira 900 tunable, pulsed Titanium:Sapphire laser system, an Olympus Fluoview 300 confocal scanning head, and a Leica DM IRE inverted microscope. It will be used in conjunction with a technique known as fluorescence correlation spectroscopy (FCS) to study intracellular protein dynamics. We will briefly explain the advantages of the two-photon system over a conventional confocal microscope, and provide some preliminary experimental results.

Intensity and amplitude correlations in the fluorescence from atoms with interacting Rydberg states

NASA Astrophysics Data System (ADS)

Xu, Qing; Mølmer, Klaus

2015-09-01

We explore the fluorescence signals from a pair of atoms driven towards Rydberg states on a three-level ladder transition. The dipole-dipole interactions between Rydberg excited atoms significantly distort the dark state and electromagnetically induced transparency behavior observed with independent atoms and, thus, their steady-state light emission. We calculate and analyze the temporal correlations between intensities and amplitudes of the signals emitted by the atoms and explain their origin in the atomic Rydberg state interactions.

Noninvasive Measurement of Bacterial Intracellular pH on a Single-Cell Level with Green Fluorescent Protein and Fluorescence Ratio Imaging Microscopy

PubMed Central

Olsen, Katja N.; Budde, Birgitte B.; Siegumfeldt, Henrik; Rechinger, K. Björn; Jakobsen, Mogens; Ingmer, Hanne

2002-01-01

We show that a pH-sensitive derivative of the green fluorescent protein, designated ratiometric GFP, can be used to measure intracellular pH (pHi) in both gram-positive and gram-negative bacterial cells. In cells expressing ratiometric GFP, the excitation ratio (fluorescence intensity at 410 and 430 nm) is correlated to the pHi, allowing fast and noninvasive determination of pHi that is ideally suited for direct analysis of individual bacterial cells present in complex environments. PMID:12147523

Recent advances in live cell imaging of hepatoma cells

PubMed Central

2014-01-01

Live cell imaging enables the study of dynamic processes of living cells in real time by use of suitable reporter proteins and the staining of specific cellular structures and/or organelles. With the availability of advanced optical devices and improved cell culture protocols it has become a rapidly growing research methodology. The success of this technique relies mainly on the selection of suitable reporter proteins, construction of recombinant plasmids possessing cell type specific promoters as well as reliable methods of gene transfer. This review aims to provide an overview of the recent developments in the field of marker proteins (bioluminescence and fluorescent) and methodologies (fluorescent resonance energy transfer, fluorescent recovery after photobleaching and proximity ligation assay) employed as to achieve an improved imaging of biological processes in hepatoma cells. Moreover, different expression systems of marker proteins and the modes of gene transfer are discussed with emphasis on the study of lipid droplet formation in hepatocytes as an example. PMID:25005127

Fluorescent probes and bioimaging: alkali metals, alkaline earth metals and pH.

PubMed

Yin, Jun; Hu, Ying; Yoon, Juyoung

2015-07-21

All living species and life forms have an absolute requirement for bio-functional metals and acid-base equilibrium chemistry owing to the critical roles they play in biological processes. Hence, a great need exists for efficient methods to detect and monitor biometals and acids. In the last few years, great attention has been paid to the development of organic molecule based fluorescent chemosensors. The availability of new synthetic fluorescent probes has made fluorescence microscopy an indispensable tool for tracing biologically important molecules and in the area of clinical diagnostics. This review highlights the recent advances that have been made in the design and bioimaging applications of fluorescent probes for alkali metals and alkaline earth metal cations, including lithium, sodium and potassium, magnesium and calcium, and for pH determination within biological systems.

The correlation of the maximum intensity of fluorescence with pigment characteristics of leaves of Betula pendula

NASA Astrophysics Data System (ADS)

Zavoruev, V. V.; Zavorueva, E. N.

2015-11-01

Using fluorimeter Junior PAM (Heinz Walz GmbH, Germany) the fluorescence parameters of leaves of Betula pendula are investigated. A linear dependence of the maximum fluorescence (Fm) of leaves from the ratio of total chlorophylls concentration to concentration of carotenoids is obtained. Such dependence is found for samples collected during the period of vegetation and for simultaneous selection of colored leaves.

Fluorescent single-digit detonation nanodiamond for biomedical applications

NASA Astrophysics Data System (ADS)

Nunn, Nicholas; d’Amora, Marta; Prabhakar, Neeraj; Panich, Alexander M.; Froumin, Natalya; Torelli, Marco D.; Vlasov, Igor; Reineck, Philipp; Gibson, Brant; Rosenholm, Jessica M.; Giordani, Silvia; Shenderova, Olga

2018-07-01

Detonation nanodiamonds (DNDs) have emerged as promising candidates for a variety of biomedical applications, thanks to different physicochemical and biological properties, such as small size and reactive surfaces. In this study, we propose carbon dot decorated single digit (4–5 nm diameter) primary particles of detonation nanodiamond as promising fluorescent probes. Due to their intrinsic fluorescence originating from tiny (1–2 atomic layer thickness) carbonaceous structures on their surfaces, they exhibit brightness suitable for in vitro imaging. Moreover, this material offers a unique, cost effective alternative to sub-10 nm nanodiamonds containing fluorescent nitrogen-vacancy color centers, which have not yet been produced at large scale. In this paper, carbon dot decorated nanodiamonds are characterized by several analytical techniques. In addition, the efficient cellular uptake and fluorescence of these particles are observed in vitro on MDA-MD-231 breast cancer cells by means of confocal imaging. Finally, the in vivo biocompatibility of carbon dot decorated nanodiamonds is demonstrated in zebrafish during the development. Our results indicate the potential of single-digit detonation nanodiamonds as biocompatible fluorescent probes. This unique material will find application in correlative light and electron microscopy, where small sized NDs can be attached to antibodies to act as a suitable dual marker for intracellular correlative microscopy of biomolecules.

Laser-Induced Fluorescence (LIF) from plant foliage

NASA Technical Reports Server (NTRS)

Chappelle, E. W.; Williams, D. L.

1986-01-01

The fluorescence spectra and fluorescence induction kinetics of green plants excited at 337 nm by a laser were studied. They correlate with plant type, as well as with changes in the physiology of the plant as the result of stress. The plant types studied include herbaceous dicots, monocots, hardwoods, conifers, and algae. These plant types could be identified on the basis of differences in either the number of fluorescent bands or the relative intensity of the bands. Differences in fluorescent spectra which could be related to vigor status are observed in conifers located in an area of high atmospheric deposition. Changes in the fluorescence spectra and induction kinetics are also seen in plants grown under conditions of nutrient deficiency and drought stress.

Laser-Induced Fluorescence (LIF) from plant foliage

NASA Technical Reports Server (NTRS)

Chappelle, Emmett W.; Williams, Darrel L.

1987-01-01

The fluorescence spectra and fluorescence induction kinetics of green plants excited at 337 nm by a laser were studied. They correlate with plant type, as well as with changes in the physiology of the plant as the result of stress. The plant types studied include herbaceous dicots, monocots, hardwoods, conifers, and algae. These plant types could be identified on the basis of differences in either the number of fluorescent bands or the relative intensity of the bands. Differences in fluorescent spectra which could be related to vigor status are observed in conifers located in an area of high atmospheric deposition. Changes in the fluorescence spectra and induction kinetics are also seen in plants grown under conditions of nutrient deficiency and drought stress.

Protein oligomerization monitored by fluorescence fluctuation spectroscopy: Self-assembly of Rubisco activase

USDA-ARS?s Scientific Manuscript database

A methodology is presented to characterize complex protein assembly pathways by fluorescence correlation spectroscopy. We have derived the total autocorrelation function describing the behavior of mixtures of labeled and unlabeled protein under equilibrium conditions. Our modeling approach allows us...

Deep Space Gateway Ecosystem Observatory

NASA Astrophysics Data System (ADS)

Huemmrich, K. F.; Campbell, P. E.; Middleton, E. M.

2018-02-01

Advance global understanding of seasonal change and diurnal variability of terrestrial ecosystem function, photosynthesis, and stress responses using spectral reflectance, thermal, and fluorescence signals.

Laser induced fluorescence technique for detecting organic matter in East China Sea

NASA Astrophysics Data System (ADS)

Chen, Peng; Wang, Tianyu; Pan, Delu; Huang, Haiqing

2017-10-01

A laser induced fluorescence (LIF) technique for fast diagnosing chromophoric dissolved organic matter (CDOM) in water is discussed. We have developed a new field-portable laser fluorometer for rapid fluorescence measurements. In addtion, the fluorescence spectral characteristics of fluorescent constituents (e.g., CDOM, chlorophyll-a) were analyzed with a spectral deconvolution method of bi-Gaussian peak function. In situ measurements by the LIF technique compared well with values measured by conventional spectrophotometer method in laboratory. A significant correlation (R2 = 0.93) was observed between fluorescence by the technique and absorption by laboratory spectrophotometer. Influence of temperature variation on LIF measurement was investigated in lab and a temperature coefficient was deduced for fluorescence correction. Distributions of CDOM fluorescence measured using this technique in the East China Sea coast were presented. The in situ result demonstrated the utility of the LIF technique for rapid detecting dissolved organic matter.

SEROLOGICAL TYPING OF STAPHYLOCOCCI BY MEANS OF FLUORESCENT ANTIBODIES I.

PubMed Central

Cohen, Jay O.; Oeding, Per

1962-01-01

Cohen, Jay O. (Communicable Disease Center, Atlanta, Ga.) and Per Oeding. Serological typing of staphylococci by means of fluorescent antibodies. I. Development of specific reagents for seven serological factors. J. Bacteriol. 84:735–741. 1962—Fluorescent antibody reagents for identifying seven antigenic factors of staphylococci have been prepared. The fluorescent staining reactions of these reagents were compared to the agglutination reactions with diagnostic cultures of coagulase-positive staphylococci. Correlation between the two serological tests was almost complete with factors a, b, i, and k. The c fluorescent antibody reagent had a somewhat broader spectrum of activity than the corresponding agglutination serum, whereas the m fluorescent antibody reagent stained fewer strains than were agglutinated in m serum. The fluorescent antibody reagent for h factor stained strains possessing h1 factor but not strains possessing only h2 factor. Fluorescent antibody reagents for specific staphylococcal factors did not stain strains of group A streptococci. PMID:14022057

Center for Advanced Propulsion Systems

DTIC Science & Technology

1993-02-01

breakup model for two chamber pressures. 7.5.4 Exciplex images for a single main injection. Images are 126 ensemble averaged for 8 individual images. Times...to obtain data using an electronic fuel injector (UCORS). Exciplex fluorescence and photographic imaging were used to study liquid and vapor...later paper, (Bower and Foster, 1993) in the same combustion bomb, the authors applied Exciplex fluorescence techniques to visualize fuel liquid and fuel

Chromosome aberrations detected by conventional karyotyping using novel mitogens in chronic lymphocytic leukemia with "normal" FISH: correlations with clinicobiologic parameters.

PubMed

Rigolin, Gian Matteo; Cibien, Francesca; Martinelli, Sara; Formigaro, Luca; Rizzotto, Lara; Tammiso, Elisa; Saccenti, Elena; Bardi, Antonella; Cavazzini, Francesco; Ciccone, Maria; Nichele, Ilaria; Pizzolo, Giovanni; Zaja, Francesco; Fanin, Renato; Galieni, Piero; Dalsass, Alessia; Mestichelli, Francesca; Testa, Nicoletta; Negrini, Massimo; Cuneo, Antonio

2012-03-08

It is unclear whether karyotype aberrations that occur in regions uncovered by the standard fluorescence in situ hybridization (FISH) panel have prognostic relevance in chronic lymphocytic leukemia (CLL). We evaluated the significance of karyotypic aberrations in a learning cohort (LC; n = 64) and a validation cohort (VC; n = 84) of patients with chronic lymphocytic leukemia with "normal" FISH. An abnormal karyotype was found in 21.5% and 35.7% of cases in the LC and VC, respectively, and was associated with a lower immunophenotypic score (P = .030 in the LC, P = .035 in the VC), advanced stage (P = .040 in the VC), and need for treatment (P = .002 in the LC, P = < .0001 in the VC). The abnormal karyotype correlated with shorter time to first treatment and shorter survival in both the LC and the VC, representing the strongest prognostic parameter. In patients with chronic lymphocytic leukemia with normal FISH, karyotypic aberrations by conventional cytogenetics with novel mitogens identify a subset of cases with adverse prognostic features.

Advances in the microrheology of complex fluids

NASA Astrophysics Data System (ADS)

Waigh, Thomas Andrew

2016-07-01

New developments in the microrheology of complex fluids are considered. Firstly the requirements for a simple modern particle tracking microrheology experiment are introduced, the error analysis methods associated with it and the mathematical techniques required to calculate the linear viscoelasticity. Progress in microrheology instrumentation is then described with respect to detectors, light sources, colloidal probes, magnetic tweezers, optical tweezers, diffusing wave spectroscopy, optical coherence tomography, fluorescence correlation spectroscopy, elastic- and quasi-elastic scattering techniques, 3D tracking, single molecule methods, modern microscopy methods and microfluidics. New theoretical techniques are also reviewed such as Bayesian analysis, oversampling, inversion techniques, alternative statistical tools for tracks (angular correlations, first passage probabilities, the kurtosis, motor protein step segmentation etc), issues in micro/macro rheological agreement and two particle methodologies. Applications where microrheology has begun to make some impact are also considered including semi-flexible polymers, gels, microorganism biofilms, intracellular methods, high frequency viscoelasticity, comb polymers, active motile fluids, blood clots, colloids, granular materials, polymers, liquid crystals and foods. Two large emergent areas of microrheology, non-linear microrheology and surface microrheology are also discussed.

Size distribution of extracellular vesicles by optical correlation techniques.

PubMed

Montis, Costanza; Zendrini, Andrea; Valle, Francesco; Busatto, Sara; Paolini, Lucia; Radeghieri, Annalisa; Salvatore, Annalisa; Berti, Debora; Bergese, Paolo

2017-10-01

Understanding the colloidal properties of extracellular vesicles (EVs) is key to advance fundamental knowledge in this field and to develop effective EV-based diagnostics, therapeutics and devices. Determination of size distribution and of colloidal stability of purified EVs resuspended in buffered media is a complex and challenging issue - because of the wide range of EV diameters (from 30 to 2000nm), concentrations of interest and membrane properties, and the possible presence of co-isolated contaminants with similar size and densities, such as protein aggregates and fat globules - which is still waiting to be fully addressed. We report here a fully detailed protocol for accurate and robust determination of the size distribution and stability of EV samples which leverages a dedicated combination of Fluorescence Correlation Spectroscopy (FCS) and Dynamic Light Scattering (DLS). The theoretical background, critical experimental steps and data analysis procedures are thoroughly presented and finally illustrated through the representative case study of EV formulations obtained from culture media of B16 melanoma cells, a murine tumor cell line used as a model for human skin cancers. Copyright © 2017 Elsevier B.V. All rights reserved.

Correlation of conformational heterogeneity of the tryptophyl side chain and time-resolved fluorescence intensity decay kinetics

NASA Astrophysics Data System (ADS)

Laws, William R.; Ross, J. B. Alexander

1992-04-01

The time-resolved fluorescence properties of a tryptophan residue should be useful for probing protein structure, function, and dynamics. To date, however, the non-single exponential fluorescence intensity decay kinetics for numerous peptides and proteins having a single tryptophan residue have not been adequately explained. Many possibilities have been considered and include: (1) contributions from the 1La and 1Lb states of indole; (2) excited-state hydrogen exchange; and (3) environmental heterogeneity from (chi) 1 and (chi) 2 rotamers. In addition, it has been suggested that generally many factors contribute to the decay and a distribution of probabilities may be more appropriate. Two recent results support multiple species due to conformational heterogeneity as the major contributor to complex kinetics. First, a rotationally constrained tryptophan analogue has fluorescence intensity decay kinetics that can be described by the sum of two exponentials with amplitudes comparable to the relative populations of the two rotational isomers. Second, the multiple exponentials observed for tyrosine-containing model compounds and peptides correlate with the (chi) 1 rotamer populations independently determined by 1H NMR. We now report similar correlations between rotamer populations and fluorescence intensity decay kinetics for a tryptophan analogue of oxytocin. It appears for this compound that either (chi) 2 rotations do not appreciably alter the indole environment, (chi) 2 rotations are rapid enough to average the observed dependence, or only one of two possible (chi) 2 populations is associated with each (chi) 1 rotamer.

Studying the Stoichiometry of Epidermal Growth Factor Receptor in Intact Cells using Correlative Microscopy.

PubMed

Peckys, Diana B; de Jonge, Niels

2015-09-11

This protocol describes the labeling of epidermal growth factor receptor (EGFR) on COS7 fibroblast cells, and subsequent correlative fluorescence microscopy and environmental scanning electron microscopy (ESEM) of whole cells in hydrated state. Fluorescent quantum dots (QDs) were coupled to EGFR via a two-step labeling protocol, providing an efficient and specific protein labeling, while avoiding label-induced clustering of the receptor. Fluorescence microscopy provided overview images of the cellular locations of the EGFR. The scanning transmission electron microscopy (STEM) detector was used to detect the QD labels with nanoscale resolution. The resulting correlative images provide data of the cellular EGFR distribution, and the stoichiometry at the single molecular level in the natural context of the hydrated intact cell. ESEM-STEM images revealed the receptor to be present as monomer, as homodimer, and in small clusters. Labeling with two different QDs, i.e., one emitting at 655 nm and at 800 revealed similar characteristic results.

Phospholipid ether analogs for the detection of colorectal tumors.

PubMed

Deming, Dustin A; Maher, Molly E; Leystra, Alyssa A; Grudzinski, Joseph P; Clipson, Linda; Albrecht, Dawn M; Washington, Mary Kay; Matkowskyj, Kristina A; Hall, Lance T; Lubner, Sam J; Weichert, Jamey P; Halberg, Richard B

2014-01-01

The treatment of localized colorectal cancer (CRC) depends on resection of the primary tumor with adequate margins and sufficient lymph node sampling. A novel imaging agent that accumulates in CRCs and the associated lymph nodes is needed. Cellectar Biosciences has developed a phospholipid ether analog platform that is both diagnostic and therapeutic. CLR1502 is a near-infrared fluorescent molecule, whereas 124/131I-CLR1404 is under clinical investigation as a PET tracer/therapeutic agent imaged by SPECT. We investigated the use of CLR1502 for the detection of intestinal cancers in a murine model and 131I-CLR1404 in a patient with metastatic CRC. Mice that develop multiple intestinal tumors ranging from adenomas to locally advanced adenocarcinomas were utilized. After 96 hours post CLR1502 injection, the intestinal tumors were analyzed using a Spectrum IVIS (Perkin Elmer) and a Fluobeam (Fluoptics). The intensity of the fluorescent signal was correlated with the histological characteristics for each tumor. Colon adenocarcinomas demonstrated increased accumulation of CLR1502 compared to non-invasive lesions (total radiant efficiency: 1.76×10(10) vs 3.27×10(9) respectively, p = 0.006). Metastatic mesenteric tumors and uninvolved lymph nodes were detected with CLR1502. In addition, SPECT imaging with 131I-CLR1404 was performed as part of a clinical trial in patients with advanced solid tumors. 131I-CLR1404 was shown to accumulate in metastatic tumors in a patient with colorectal adenocarcinoma. Together, these compounds might enhance our ability to properly resect CRCs through better localization of the primary tumor and improved lymph node identification as well as detect distant disease.

Phospholipid Ether Analogs for the Detection of Colorectal Tumors

PubMed Central

Deming, Dustin A.; Maher, Molly E.; Leystra, Alyssa A.; Grudzinski, Joseph P.; Clipson, Linda; Albrecht, Dawn M.; Washington, Mary Kay; Matkowskyj, Kristina A.; Hall, Lance T.; Lubner, Sam J.; Weichert, Jamey P.; Halberg, Richard B.

2014-01-01

The treatment of localized colorectal cancer (CRC) depends on resection of the primary tumor with adequate margins and sufficient lymph node sampling. A novel imaging agent that accumulates in CRCs and the associated lymph nodes is needed. Cellectar Biosciences has developed a phospholipid ether analog platform that is both diagnostic and therapeutic. CLR1502 is a near-infrared fluorescent molecule, whereas 124/131I-CLR1404 is under clinical investigation as a PET tracer/therapeutic agent imaged by SPECT. We investigated the use of CLR1502 for the detection of intestinal cancers in a murine model and 131I-CLR1404 in a patient with metastatic CRC. Mice that develop multiple intestinal tumors ranging from adenomas to locally advanced adenocarcinomas were utilized. After 96 hours post CLR1502 injection, the intestinal tumors were analyzed using a Spectrum IVIS (Perkin Elmer) and a Fluobeam (Fluoptics). The intensity of the fluorescent signal was correlated with the histological characteristics for each tumor. Colon adenocarcinomas demonstrated increased accumulation of CLR1502 compared to non-invasive lesions (total radiant efficiency: 1.76×1010 vs 3.27×109 respectively, p = 0.006). Metastatic mesenteric tumors and uninvolved lymph nodes were detected with CLR1502. In addition, SPECT imaging with 131I-CLR1404 was performed as part of a clinical trial in patients with advanced solid tumors. 131I-CLR1404 was shown to accumulate in metastatic tumors in a patient with colorectal adenocarcinoma. Together, these compounds might enhance our ability to properly resect CRCs through better localization of the primary tumor and improved lymph node identification as well as detect distant disease. PMID:25286226

Fluorescence correlation spectroscopy analysis for accurate determination of proportion of doubly labeled DNA in fluorescent DNA pool for quantitative biochemical assays.

PubMed

Hou, Sen; Sun, Lili; Wieczorek, Stefan A; Kalwarczyk, Tomasz; Kaminski, Tomasz S; Holyst, Robert

2014-01-15

Fluorescent double-stranded DNA (dsDNA) molecules labeled at both ends are commonly produced by annealing of complementary single-stranded DNA (ssDNA) molecules, labeled with fluorescent dyes at the same (3' or 5') end. Because the labeling efficiency of ssDNA is smaller than 100%, the resulting dsDNA have two, one or are without a dye. Existing methods are insufficient to measure the percentage of the doubly-labeled dsDNA component in the fluorescent DNA sample and it is even difficult to distinguish the doubly-labeled DNA component from the singly-labeled component. Accurate measurement of the percentage of such doubly labeled dsDNA component is a critical prerequisite for quantitative biochemical measurements, which has puzzled scientists for decades. We established a fluorescence correlation spectroscopy (FCS) system to measure the percentage of doubly labeled dsDNA (PDL) in the total fluorescent dsDNA pool. The method is based on comparative analysis of the given sample and a reference dsDNA sample prepared by adding certain amount of unlabeled ssDNA into the original ssDNA solution. From FCS autocorrelation functions, we obtain the number of fluorescent dsDNA molecules in the focal volume of the confocal microscope and PDL. We also calculate the labeling efficiency of ssDNA. The method requires minimal amount of material. The samples have the concentration of DNA in the nano-molar/L range and the volume of tens of microliters. We verify our method by using restriction enzyme Hind III to cleave the fluorescent dsDNA. The kinetics of the reaction depends strongly on PDL, a critical parameter for quantitative biochemical measurements. Copyright © 2013 Elsevier B.V. All rights reserved.

Confocal fluorescence techniques in industrial application

NASA Astrophysics Data System (ADS)

Eggeling, Christian; Gall, Karsten; Palo, Kaupo; Kask, Peet; Brand, Leif

2003-06-01

The FCS+plus family of evaluation tools for confocal fluorescence spectroscopy, which was developed during recent years, offers a comprehensive view to a series of fluorescence properties. Originating in fluorescence correlation spectroscopy (FCS) and using similar experimental equipment, a system of signal processing methods such as fluorescence intensity distribution analysis (FIDA) was created to analyze in detail the fluctuation behavior of fluorescent particles within a small area of detection. Giving simultaneous access to molecular parameters like concentration, translational and rotational diffusion, molecular brightness, and multicolor coincidence, this portfolio was enhanced by more traditional techniques of fluorescence lifetime as well as time-resolved anisotropy determination. The cornerstones of the FCS+plus methodology will be shortly described. The inhibition of a phosphatase enzyme activity gives a comprehensive industrial application that demonstrates FCS+plus' versatility and its potential for pharmaceutical drug discovery.

Quantitative single-molecule imaging by confocal laser scanning microscopy.

PubMed

Vukojevic, Vladana; Heidkamp, Marcus; Ming, Yu; Johansson, Björn; Terenius, Lars; Rigler, Rudolf

2008-11-25

A new approach to quantitative single-molecule imaging by confocal laser scanning microscopy (CLSM) is presented. It relies on fluorescence intensity distribution to analyze the molecular occurrence statistics captured by digital imaging and enables direct determination of the number of fluorescent molecules and their diffusion rates without resorting to temporal or spatial autocorrelation analyses. Digital images of fluorescent molecules were recorded by using fast scanning and avalanche photodiode detectors. In this way the signal-to-background ratio was significantly improved, enabling direct quantitative imaging by CLSM. The potential of the proposed approach is demonstrated by using standard solutions of fluorescent dyes, fluorescently labeled DNA molecules, quantum dots, and the Enhanced Green Fluorescent Protein in solution and in live cells. The method was verified by using fluorescence correlation spectroscopy. The relevance for biological applications, in particular, for live cell imaging, is discussed.

Detection of Subclinical Arthritis in Mice by a Thrombin Receptor-Derived Imaging Agent.

PubMed

Friedman, Beth; Whitney, Michael A; Savariar, Elamprakash N; Caneda, Christa; Steinbach, Paul; Xiong, Qing; Hingorani, Dina V; Crisp, Jessica; Adams, Stephen R; Kenner, Michael; Lippert, Csilla N; Nguyen, Quyen T; Guma, Monica; Tsien, Roger Y; Corr, Maripat

2018-01-01

Functional imaging of synovitis could improve both early detection of rheumatoid arthritis (RA) and long-term outcomes. Given the intersection of inflammation with coagulation protease activation, this study was undertaken to examine coagulation protease activities in arthritic mice with a dual-fluorescence ratiometric activatable cell-penetrating peptide (RACPP) that has a linker, norleucine (Nle)-TPRSFL, with a cleavage site for thrombin. K/BxN-transgenic mice with chronic arthritis and mice with day 1 passive serum-transfer arthritis were imaged in vivo for Cy5:Cy7 emission ratiometric fluorescence from proteolytic cleavage and activation of RACPP NleTPRSFL . Joint thickness in mice with serum-transfer arthritis was measured from days 0 to 10. The cleavage-evoked release of Cy5-tagged tissue-adhesive fragments enabled microscopic correlation with immunohistochemistry for inflammatory markers. Thrombin dependence of ratiometric fluorescence was tested by ex vivo application of RACPP NleTPRSFL and argatroban to cryosections obtained from mouse hind paws on day 1 of serum-transfer arthritis. In chronic arthritis, RACPP NleTPRSFL fluorescence ratios of Cy5:Cy7 emission were significantly higher in diseased swollen ankles of K/BxN-transgenic mice than in normal mouse ankles. A high ratio of RACPP NleTPRSFL fluorescence in mouse ankles and toes on day 1 of serum-transfer arthritis correlated with subsequent joint swelling. Foci of high ratiometric fluorescence localized to inflammation, as demarcated by immune reactivity for citrullinated histones, macrophages, mast cells, and neutrophils, in soft tissue on day 1 of serum-transfer arthritis. Ex vivo application of RACPP NleTPRSFL to cryosections obtained from mice on day 1 of serum-transfer arthritis produced ratiometric fluorescence that was inhibited by argatroban. RACPP NleTPRSFL activation detects established experimental arthritis, and the detection of inflammation by RACPP NleTPRSFL on day 1 of serum-transfer arthritis correlates with disease progression. © 2017, American College of Rheumatology.

Selective Chemical Labeling of Proteins with Small Fluorescent Molecules Based on Metal-Chelation Methodology

PubMed Central

Soh, Nobuaki

2008-01-01

Site-specific chemical labeling utilizing small fluorescent molecules is a powerful and attractive technique for in vivo and in vitro analysis of cellular proteins, which can circumvent some problems in genetic encoding labeling by large fluorescent proteins. In particular, affinity labeling based on metal-chelation, advantageous due to the high selectivity/simplicity and the small tag-size, is promising, as well as enzymatic covalent labeling, thereby a variety of novel methods have been studied in recent years. This review describes the advances in chemical labeling of proteins, especially highlighting the metal-chelation methodology. PMID:27879749

Advanced industrial fluorescence metrology used for qualification of high quality optical materials

NASA Astrophysics Data System (ADS)

Engel, Axel; Becker, Hans-Juergen; Sohr, Oliver; Haspel, Rainer; Rupertus, Volker

2003-11-01

Schott Glas is developing and producing the optical material for various specialized applications in telecommunication, biomedical, optical, and micro lithography technology. The requirements on quality for optical materials are extremely high and still increasing. For example in micro lithography applications the impurities of the material are specified to be in the low ppb range. Usually the impurities in the lower ppb range are determined using analytical methods like LA ICP-MS and Neutron Activation Analysis. On the other hand absorption and laser resistivity of optical material is qualified with optical methods like precision spectral photometers and in-situ transmission measurements having UV lasers. Analytical methods have the drawback that they are time consuming and rather expensive, whereas the sensitivity for the absorption method will not be sufficient to characterize the future needs (coefficient much below 10-3 cm-1). In order to achieve the current and future quality requirements a Jobin Yvon FLUOROLOG 3.22 fluorescence spectrometer is employed to enable fast and precise qualification and analysis. The main advantage of this setup is the combination of highest sensitivity (more than one order of magnitude higher sensitivity that state of the art UV absorption spectroscopy) and fast measurement and evaluation cycles (several minutes compared to several hours necessary for chemical analytics). An overview is given for spectral characteristics and using specified standards. Moreover correlations to the material qualities are shown. In particular we have investigated the elementary fluorescence and absorption of rare earth element impurities as well as defects induced luminescence originated by impurities.

Correlation of cholinergic drug induced quenching of acetylcholinesterase bound thioflavin-T fluorescence with their inhibition activity

NASA Astrophysics Data System (ADS)

Islam, Mullah Muhaiminul; Rohman, Mostofa Ataur; Gurung, Arun Bahadur; Bhattacharjee, Atanu; Aguan, Kripamoy; Mitra, Sivaprasad

2018-01-01

The development of new acetylcholinesterase inhibitors (AChEIs) and subsequent assay of their inhibition efficiency is considered to be a key step for AD treatment. The fluorescence intensity of thioflavin-T (ThT) bound in the active site of acetylcholinesterase (AChE) quenches substantially in presence of standard AChEI drugs due to the dynamic replacement of the fluorophore from the AChE active site as confirmed from steady state emission as well as time-resolved fluorescence anisotropy measurement and molecular dynamics simulation in conjunction with docking calculation. The parametrized % quenching data for individual system shows excellent correlation with enzyme inhibition activity measured independently by standard Ellman AChE assay method in a high throughput plate reader system. The results are encouraging towards design of a fluorescence intensity based AChE inhibition assay method and may provide a better toolset to rapidly evaluate as well as develop newer AChE-inhibitors for AD treatment.

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.

Peculiarities of the statistics of spectrally selected fluorescence radiation in laser-pumped dye-doped random media

NASA Astrophysics Data System (ADS)

Yuvchenko, S. A.; Ushakova, E. V.; Pavlova, M. V.; Alonova, M. V.; Zimnyakov, D. A.

2018-04-01

We consider the practical realization of a new optical probe method of the random media which is defined as the reference-free path length interferometry with the intensity moments analysis. A peculiarity in the statistics of the spectrally selected fluorescence radiation in laser-pumped dye-doped random medium is discussed. Previously established correlations between the second- and the third-order moments of the intensity fluctuations in the random interference patterns, the coherence function of the probe radiation, and the path difference probability density for the interfering partial waves in the medium are confirmed. The correlations were verified using the statistical analysis of the spectrally selected fluorescence radiation emitted by a laser-pumped dye-doped random medium. Water solution of Rhodamine 6G was applied as the doping fluorescent agent for the ensembles of the densely packed silica grains, which were pumped by the 532 nm radiation of a solid state laser. The spectrum of the mean path length for a random medium was reconstructed.

Community detection for fluorescent lifetime microscopy image segmentation

NASA Astrophysics Data System (ADS)

Hu, Dandan; Sarder, Pinaki; Ronhovde, Peter; Achilefu, Samuel; Nussinov, Zohar

2014-03-01

Multiresolution community detection (CD) method has been suggested in a recent work as an efficient method for performing unsupervised segmentation of fluorescence lifetime (FLT) images of live cell images containing fluorescent molecular probes.1 In the current paper, we further explore this method in FLT images of ex vivo tissue slices. The image processing problem is framed as identifying clusters with respective average FLTs against a background or "solvent" in FLT imaging microscopy (FLIM) images derived using NIR fluorescent dyes. We have identified significant multiresolution structures using replica correlations in these images, where such correlations are manifested by information theoretic overlaps of the independent solutions ("replicas") attained using the multiresolution CD method from different starting points. In this paper, our method is found to be more efficient than a current state-of-the-art image segmentation method based on mixture of Gaussian distributions. It offers more than 1:25 times diversity based on Shannon index than the latter method, in selecting clusters with distinct average FLTs in NIR FLIM images.

Real-time visualization of oxidative stress in a floating macrophyte Lemna minor L. exposed to cadmium, copper, menadione, and AAPH.

PubMed

Razinger, Jaka; Drinovec, Luka; Zrimec, Alexis

2010-12-01

An ultra-sensitive digital imaging system was employed to visualize oxidative stress in intact L. minor plants exposed to Cd, Cu, menadione, AAPH, and ascorbate in real time. The increase of ROS production was assessed by measuring the rate of fluorescence intensity increases of the test medium supplemented with a fluorescing probe (dichlorofluorescein diacetate). The addition of 100 μM CdCl₂ or 100 μM CuSO₄ to the growth medium resulted in a significant increase of medium fluorescence. Additionally, CuSO₄ caused a significantly higher fluorescence intensity than CdCl₂ did. A strong positive correlation (R² = 0.99) between menadione concentration and fluorescence intensity was observed. The positive correlation between AAPH concentration and fluorescence intensity was not as strong as in the case of menadione (R² = 0.81). Menadione induced a stronger oxidative stress than similar concentration of AAPH. The addition of 100 μM ascorbate to L. minor treated with 50 μM menadione significantly reduced the fluorescence intensity increase. A linear trend of the fluorescence increase was observed in all treatments, indicating that chemical-induced oxidative stress is a gradual process and that the applied concentrations of the chemicals caused a constant increased production of ROS with different intensities, depending on the treatment. This is the combined result of a gradual diminishing of antioxidant reserves and accumulating oxidative damage. The observed rates of ROS production were slower than those in the studies using cell cultures. Copyright © 2009 Wiley Periodicals, Inc.

[Study on optical characteristics of chromophoric dissolved organic matter (CDOM) in rainwater by fluorescence excitation-emission matrix and absorbance spectroscopy].

PubMed

Cheng, Yuan-yue; Guo, Wei-dong; Long, Ai-min; Chen, Shao-yong

2010-09-01

The optical characteristics of chromophoric dissolved organic matter (CDOM) were determined in rain samples collected in Xiamen Island, during a rainy season in 2007, using fluorescence excitation-emission matrix spectroscopy associated with UV-Vis absorbance spectra. Results showed that the absorbance spectra of CDOM in rain samples decreased exponentially with wavelength. The absorbance coefficient at 300 nm [a(300)] ranged from 0.27 to 3.45 m(-1), which would be used as an index of CDOM abundance, and the mean value was 1.08 m(-1). The content of earlier stage of precipitation events was higher than that of later stage of precipitation events, which implied that anthropogenic sources or atmospheric pollution or air mass types were important contributors to CDOM levels in precipitation. EEMs spectra showed 4 types of fluorescence signals (2 humic-like fluorescence peaks and 2 protein-like fluorescence peaks) in rainwater samples, and there were significant positive correlations of peak A with C and peak B with S, showing their same sources or some relationship of the two humic-like substance and the two protein-like substance. The strong positive correlations of the two humic-like fluorescence peaks with a(300), suggested that the chromophores responsible for absorbance might be the same as fluorophores responsible for fluorescence. Results showed that the presence of highly absorbing and fluorescing CDOM in rainwater is of significant importance in atmospheric chemistry and might play a previously unrecognized role in the wavelength dependent spectral attenuation of solar radiation by atmospheric waters.

δ-aminolevulinic acid–induced protoporphyrin IX concentration correlates with histopathologic markers of malignancy in human gliomas: the need for quantitative fluorescence-guided resection to identify regions of increasing malignancy

PubMed Central

Valdés, Pablo A.; Kim, Anthony; Brantsch, Marco; Niu, Carolyn; Moses, Ziev B.; Tosteson, Tor D.; Wilson, Brian C.; Paulsen, Keith D.; Roberts, David W.; Harris, Brent T.

2011-01-01

Extent of resection is a major goal and prognostic factor in the treatment of gliomas. In this study we evaluate whether quantitative ex vivo tissue measurements of δ-aminolevulinic acid–induced protoporphyrin IX (PpIX) identify regions of increasing malignancy in low- and high-grade gliomas beyond the capabilities of current fluorescence imaging in patients undergoing fluorescence-guided resection (FGR). Surgical specimens were collected from 133 biopsies in 23 patients and processed for ex vivo neuropathological analysis: PpIX fluorimetry to measure PpIX concentrations (CPpIX) and Ki-67 immunohistochemistry to assess tissue proliferation. Samples displaying visible levels of fluorescence showed significantly higher levels of CPpIX and tissue proliferation. CPpIX was strongly correlated with histopathological score (nonparametric) and tissue proliferation (parametric), such that increasing levels of CPpIX were identified with regions of increasing malignancy. Furthermore, a large percentage of tumor-positive biopsy sites (∼40%) that were not visibly fluorescent under the operating microscope had levels of CPpIX greater than 0.1 µg/mL, which indicates that significant PpIX accumulation exists below the detection threshold of current fluorescence imaging. Although PpIX fluorescence is recognized as a visual biomarker for neurosurgical resection guidance, these data show that it is quantitatively related at the microscopic level to increasing malignancy in both low- and high-grade gliomas. This work suggests a need for improved PpIX fluorescence detection technologies to achieve better sensitivity and quantification of PpIX in tissue during surgery. PMID:21798847

Maximizing Aggregation of Organic Fluorophores to Prolong Fluorescence Lifetime for Two-Photon Fluorescence Lifetime Imaging.

PubMed

Hu, Wenbo; Guo, Lihong; Bai, Lei; Miao, Xiaofei; Ni, Yun; Wang, Qi; Zhao, Hui; Xie, Meng; Li, Lin; Lu, Xiaomei; Huang, Wei; Fan, Quli

2018-05-28

Two-photon fluorescence lifetime imaging (TP-FLIM) not only permits imaging deep inside the tissues with precise spatial manipulation but also circumvents tissue autofluorescence, holding tremendous promise in molecular imaging. However, the serious lack of suitable contrast agents with long fluorescence lifetime and efficient two-photon absorption (TPA) greatly limits the advance of TP-FLIM. This study reports a simple approach to fabricate water-soluble organic semiconducting nanoparticles [thioxanthone (TXO) NPs] with ultralong fluorescence lifetime and efficient TPA for in vivo TP-FLIM. The approach utilizes the aggregation of a specifically selected thermally activated delayed fluorescence (TADF) fluorophore to prolong its fluorescence lifetime. Encapsulating the TADF fluorophore within an amphiphilic copolymer not only maximizes its aggregation but also obtains TXO NPs with efficient TPA. Importantly, as-prepared TXO NPs exhibit a considerably long fluorescence lifetime at a magnitude of 4.2 µs, which is almost 1000 times larger than that of existing organic contrast agents. Moreover, such long fluorescence lifetime is almost oxygen-inert, readily realizing both in vitro and in vivo TP-FLIM. This work may set valuable guidance for designing organic semiconducting materials with ultralong fluorescence lifetimes to fulfill the potential of FLIM. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Organic Dots Based on AIEgens for Two-Photon Fluorescence Bioimaging.

PubMed

Lou, Xiaoding; Zhao, Zujin; Tang, Ben Zhong

2016-12-01

Two-photon fluorescence imaging technique is a powerful bioanalytical approach in terms of high photostability, low photodamage, high spatiotemporal resolution. Recently, fluorescent organic dots comprised of organic emissive cores and a polymeric matrix are emerging as promising contrast reagents for two-photon fluorescence imaging, owing to their numerous merits of high and tunable fluorescence, good biocompatibility, strong photobleaching resistance, and multiple surface functionality. The emissive core is crucial for organic dots to get high brightness but many conventional chromophores often encounter a severe problem of fluorescence quenching when they form aggregates. To solve this problem, fluorogens featuring aggregation-induced emission (AIE) can fluoresce strongly in aggregates, and thus become ideal candidates for fluorescent organic dots. In addition, two-photon absorption property of the dots can be readily improved by just increase loading contents of AIE fluorogen (AIEgen). Hence, organic dots based on AIEgens have exhibited excellent performances in two-photon fluorescence in vitro cellular imaging, and in vivo vascular architecture visualization of mouse skin, muscle, brain and skull bone. In view of the rapid advances in this important research field, here, we highlight representative fluorescent organic dots with an emissive core of AIEgen aggregate, and discuss their great potential in bioimaging applications. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Advanced Experiments in Nuclear Science, Volume I: Advanced Nuclear Physics and Chemistry Experiments.

ERIC Educational Resources Information Center

Duggan, Jerome L.; And Others

The experiments in this manual represent state-of-the-art techniques which should be within the budgetary constraints of a college physics or chemistry department. There are fourteen experiments divided into five modules. The modules are on X-ray fluorescence, charged particle detection, neutron activation analysis, X-ray attenuation, and…

Complexation of β-cyclodextrin with dual molecular probes bearing fluorescent and paramagnetic moieties linked by short polyether chains.

PubMed

Mocanu, S; Matei, I; Ionescu, S; Tecuceanu, V; Marinescu, G; Ionita, P; Culita, D; Leonties, A; Ionita, Gabriela

2017-10-18

Electron paramagnetic resonance (EPR) and fluorescence spectroscopies provide molecular-level insights on the interaction of paramagnetic and fluorescent species with the microenvironment. A series of dual molecular probes bearing fluorescent and paramagnetic moieties linked by flexible short polyether chains have been synthesized. These new molecular probes open the possibility to investigate various multi-component systems such as host-guest systems, polymeric micelles, gels and protein solutions by using EPR and fluorescence spectroscopies concertedly. The EPR and fluorescence spectra of these compounds show that the dependence of the rotational correlation time and fluorescence quantum yield on the chain length of the linker is not linear, due to the flexibility of the polyether linker. The quenching effect of the nitroxide moiety on the fluorescence intensity of the pyrene group varies with the linker length and flexibility. The interaction of these dual molecular probes with β-cyclodextrin, in solution and in polymeric gels, was evaluated and demonstrated by analysis of EPR and fluorescence spectra.

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.

Determining Protease Activity In Vivo by Fluorescence Cross-Correlation Analysis

PubMed Central

Kohl, Tobias; Haustein, Elke; Schwille, Petra

2005-01-01

To date, most biochemical approaches to unravel protein function have focused on purified proteins in vitro. Whereas they analyze enzyme performance under assay conditions, they do not necessarily tell us what is relevant within a living cell. Ideally, cellular functions should be examined in situ. In particular, association/dissociation reactions are ubiquitous, but so far there is no standard technique permitting online analysis of these processes in vivo. Featuring single-molecule sensitivity combined with intrinsic averaging, fluorescence correlation spectroscopy is a minimally invasive technique ideally suited to monitor proteins. Moreover, endogenous fluorescence-based assays can be established by genetically encoding fusions of autofluorescent proteins and cellular proteins, thus avoiding the disadvantages of in vitro protein labeling and subsequent delivery to cells. Here, we present an in vivo protease assay as a model system: Green and red autofluorescent proteins were connected by Caspase-3- sensitive and insensitive protein linkers to create double-labeled protease substrates. Then, dual-color fluorescence cross-correlation spectroscopy was employed to study the protease reaction in situ. Allowing assessment of multiple dynamic parameters simultaneously, this method provided internal calibration and improved experimental resolution for quantifying protein stability. This approach, which is easily extended to reversible protein-protein interactions, seems very promising for elucidating intracellular protein functions. PMID:16055538

Accumulation of Maillard reaction products in skin collagen in diabetes and aging.

PubMed Central

Dyer, D G; Dunn, J A; Thorpe, S R; Bailie, K E; Lyons, T J; McCance, D R; Baynes, J W

1993-01-01

To investigate the contribution of glycation and oxidation reactions to the modification of insoluble collagen in aging and diabetes, Maillard reaction products were measured in skin collagen from 39 type 1 diabetic patients and 52 nondiabetic control subjects. Compounds studied included fructoselysine (FL), the initial glycation product, and the glycoxidation products, N epsilon-(carboxymethyl) lysine (CML) and pentosidine, formed during later Maillard reactions. Collagen-linked fluorescence was also studied. In nondiabetic subjects, glycation of collagen (FL content) increased only 33% between 20 and 85 yr of age. In contrast, CML, pentosidine and fluorescence increased five-fold, correlating strongly with age. In diabetic patients, collagen FL was increased threefold compared with nondiabetic subjects, correlating strongly with glycated hemoglobin but not with age. Collagen CML, pentosidine and fluorescence were increased up to twofold in diabetic compared with control patients: this could be explained by the increase in glycation alone, without invoking increased oxidative stress. There were strong correlations among CML, pentosidine and fluorescence in both groups, providing evidence for age-dependent chemical modification of collagen via the Maillard reaction, and acceleration of this process in diabetes. These results support the description of diabetes as a disease characterized by accelerated chemical aging of long-lived tissue proteins. PMID:8514858

Oligomeric State of Lipocalin-1 (LCN1) by Multiangle Laser Light Scattering and Fluorescence Anisotropy Decay

PubMed Central

Gasymov, Oktay K.; Abduragimov, Adil R.; Merschak, Petra; Redl, Bernhard; Glasgow, Ben J.

2007-01-01

Multiangle laser light scattering and fluorescence anisotropy decay measurements clarified the oligomeric states of native and recombinant tear lipocalin (lipocalin-1, TL). Native TL is monomeric. Recombinant TL (5-68 μM) with or without the histidine tag shows less than 7% dimer formation that is not in equilibrium with the monomeric form. Fluorescence anisotropy decay showed a correlation time of 9-10 ns for TL (10 μM- 1mM). Hydrodynamic calculations based on the crystallographic structure of a monomeric TL mutant closely concur with the observed correlation time. The solution properties calculated with HYDROPRO and SOLPRO programs from the available crystallographic structure of a monomeric TL mutant concur closely with the observed fluorescence anisotropy decay. The resulting model shows that protein topology is the major determinant of rotational correlation time and accounts for deviation from the Stokes-Einstein relation. The data challenge previous gel filtration studies to show that native TL exists predominantly as a monomer in solution rather than as a dimer. Delipidation of TL results in a formation of a complex oligomeric state (up to 25%). These findings are important as the dynamic processes in the tear film are limited by diffusional, translational as well as rotational, properties of the protein. PMID:17869594

Correlation of tryptophan fluorescence intensity decay parameters with sup 1 H NMR-determined rotamer conformations: (tryptophan sup 2 )oxytocin

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

Ross, J.B.A.; Schwartz, G.P.; Laws, W.R.

1992-02-18

While the fluorescence decay kinetics of tyrosine model compounds can be explained in terms of heterogeneity derived from the three ground-state {chi}{sup 1} rotamers, a similar correlation has yet to be directly observed for a tryptophan residue. In addition, the asymmetric indole ring might also lead to heterogeneity from {chi}{sup 2} rotations. In this paper, the time-resolved and steady-state fluorescence properties of (tryptophan{sup 2})oxytocin at pH 3 are presented and compared with {sup 1}H NMR results. According to the unrestricted analyses of individual fluorescence decay curves taken as a function of emission wavelength-independent decay constants, only three exponential terms aremore » required. In addition, the preexponential weighting factors (amplitudes) have the same relative relationship (weights) as the {sup 1}H NMR-determined {chi}{sup 1} rotamer populations of the indole side chain. {sup 15}N was used in heteronuclear coupling experiments to confirm the rotamer assignments. Inclusion of a linked function restricting the decay amplitudes to the {chi}{sup 1} rotamer populations in the individual decay curve analyses and in the global analysis confirms this correlation. According to qualitative nuclear Overhauser data, there are two {chi}{sup 2} populations.« less

Correlative Light- and Electron Microscopy Using Quantum Dot Nanoparticles.

PubMed

Killingsworth, Murray C; Bobryshev, Yuri V

2016-08-07

A method is described whereby quantum dot (QD) nanoparticles can be used for correlative immunocytochemical studies of human pathology tissue using widefield fluorescence light microscopy and transmission electron microscopy (TEM). To demonstrate the protocol we have immunolabeled ultrathin epoxy sections of human somatostatinoma tumor using a primary antibody to somatostatin, followed by a biotinylated secondary antibody and visualization with streptavidin conjugated 585 nm cadmium-selenium (CdSe) quantum dots (QDs). The sections are mounted on a TEM specimen grid then placed on a glass slide for observation by widefield fluorescence light microscopy. Light microscopy reveals 585 nm QD labeling as bright orange fluorescence forming a granular pattern within the tumor cell cytoplasm. At low to mid-range magnification by light microscopy the labeling pattern can be easily recognized and the level of non-specific or background labeling assessed. This is a critical step for subsequent interpretation of the immunolabeling pattern by TEM and evaluation of the morphological context. The same section is then blotted dry and viewed by TEM. QD probes are seen to be attached to amorphous material contained in individual secretory granules. Images are acquired from the same region of interest (ROI) seen by light microscopy for correlative analysis. Corresponding images from each modality may then be blended to overlay fluorescence data on TEM ultrastructure of the corresponding region.

Mapping the lignin distribution in pretreated sugarcane bagasse by confocal and fluorescence lifetime imaging microscopy

PubMed Central

2013-01-01

Background Delignification pretreatments of biomass and methods to assess their efficacy are crucial for biomass-to-biofuels research and technology. Here, we applied confocal and fluorescence lifetime imaging microscopy (FLIM) using one- and two-photon excitation to map the lignin distribution within bagasse fibers pretreated with acid and alkali. The evaluated spectra and decay times are correlated with previously calculated lignin fractions. We have also investigated the influence of the pretreatment on the lignin distribution in the cell wall by analyzing the changes in the fluorescence characteristics using two-photon excitation. Eucalyptus fibers were also analyzed for comparison. Results Fluorescence spectra and variations of the decay time correlate well with the delignification yield and the lignin distribution. The decay dependences are considered two-exponential, one with a rapid (τ1) and the other with a slow (τ2) decay time. The fastest decay is associated to concentrated lignin in the bagasse and has a low sensitivity to the treatment. The fluorescence decay time became longer with the increase of the alkali concentration used in the treatment, which corresponds to lignin emission in a less concentrated environment. In addition, the two-photon fluorescence spectrum is very sensitive to lignin content and accumulation in the cell wall, broadening with the acid pretreatment and narrowing with the alkali one. Heterogeneity of the pretreated cell wall was observed. Conclusions Our results reveal lignin domains with different concentration levels. The acid pretreatment caused a disorder in the arrangement of lignin and its accumulation in the external border of the cell wall. The alkali pretreatment efficiently removed lignin from the middle of the bagasse fibers, but was less effective in its removal from their surfaces. Our results evidenced a strong correlation between the decay times of the lignin fluorescence and its distribution within the cell wall. A new variety of lignin fluorescence states were accessed by two-photon excitation, which allowed an even broader, but complementary, optical characterization of lignocellulosic materials. These results suggest that the lignin arrangement in untreated bagasse fiber is based on a well-organized nanoenvironment that favors a very low level of interaction between the molecules. PMID:23548159

Comparison of five techniques for the detection of Renibacterium salmoninarum in adult coho salmon.

USGS Publications Warehouse

Pascho, R.J.; Elliott, D.G.; Mallett, R.W.; Mulcahy, D.

1987-01-01

Samples of kidney, spleen, coelomic fluid, and blood from 56 sexually mature coho salmon Oncorhynchus kisutch were examined for infection by Renibacterium salmoninarum by five methods. The overall prevalence (all sample types combined) of R. salmoninarum in the fish was 100% by the enzyme-linked immunosorbent assay, 86% by the combined results of the direct fluorescent antibody and the direct filtration-fluorescent antibody techniques, 39% by culture, 11% by counterimmunoelectrophoresis, and 5% by agarose gel immunodiffusion. There was a significant positive correlation (P < 0.001) between the enzyme-linked immunosorbent assay absorbance levels and the counts by fluorescent antibody techniques for kidney, spleen, and coelomic fluid, and significant positive correlations (P < 0.001) in enzyme-linked immunosorbent assay absorbance levels for all four of the sample types.

Fluorescence Correlation Spectroscopy to Study Diffusion of Polymer Chains within Layered Hydrogen-Bonded Polymer Films

NASA Astrophysics Data System (ADS)

Pristinski, Denis; Kharlampieva, Evguenia; Sukhishvili, Svetlana

2002-03-01

Fluorescence Correlation Spectroscopy (FCS) has been used to probe molecular motions within polymer multilayers formed by hydrogen-bonding sequential self-assembly. Polyethylene glycol (PEG) molecules were end-labeled with the fluorescent tags, and self-assembled with polymethacrylic acid (PMAA) using layer-by-layer deposition. We have found that molecules included in the top adsorbed layer have significant mobility at the millisecond time scale, probably due to translational diffusion. However, their dynamics deviate from classical Brownian motion with a single diffusion time. Possible reasons for the deviation are discussed. We found that motions were significantly slowed with increasing depth within the PEG/PMAA multilayer. This phenomena occured in a narrow pH range around 4.0 in which intermolecular interactions were relatively weak.

Reaction-based small-molecule fluorescent probes for dynamic detection of ROS and transient redox changes in living cells and small animals.

PubMed

Lü, Rui

2017-09-01

Dynamic detection of transient redox changes in living cells and animals has broad implications for human health and disease diagnosis, because intracellular redox homeostasis regulated by reactive oxygen species (ROS) plays important role in cell functions, normal physiological functions and some serious human diseases (e.g., cancer, Alzheimer's disease, diabetes, etc.) usually have close relationship with the intracellular redox status. Small-molecule ROS-responsive fluorescent probes can act as powerful tools for dynamic detection of ROS and redox changes in living cells and animals through fluorescence imaging techniques; and great advances have been achieved recently in the design and synthesis of small-molecule ROS-responsive fluorescent probes. This article highlights up-to-date achievements in designing and using the reaction-based small-molecule fluorescent probes (with high sensitivity and selectivity to ROS and redox cycles) in the dynamic detection of ROS and transient redox changes in living cells and animals through fluorescence imaging. Copyright © 2017. Published by Elsevier Ltd.

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.

Combination of Fluorescence-Guided Surgery With Photodynamic Therapy for the Treatment of Cancer

PubMed Central

He, Jun; Yang, Leping; Yi, Wenjun; Fan, Wentao; Wen, Yu; Miao, Xiongying; Xiong, Li

2017-01-01

Specific visualization of body parts is needed during surgery. Fluorescence-guided surgery (FGS) uses a fluorescence contrast agent for in vivo tumor imaging to detect and identify both malignant and normal tissues. There are several advantages and clinical benefits of FGS over other conventional medical imaging modalities, such as its safety, effectiveness, and suitability for real-time imaging in the operating room. Recent advancements in contrast agents and intraoperative fluorescence imaging devices have led to a greater potential for intraoperative fluorescence imaging in clinical applications. Photodynamic therapy (PDT) is an alternative modality to treat tumors, which uses a light-sensitive drug (photosensitizers) and special light to destroy the targeted tissues. In this review, we discuss the fluorescent contrast agents, some newly developed imaging devices, and the successful clinical application of FGS. Additionally, we present the combined strategy of FGS with PDT to further improve the therapeutic effect for patients with cancer. Taken together, this review provides a unique perspective and summarization of FGS. PMID:28849712

Single-Walled Carbon Nanotubes as Fluorescence Biosensors for Pathogen Recognition in Water Systems

DOE PAGES

Upadhyayula, Venkata K. K.; Ghoshroy, Soumitra; Nair, Vinod S.; ...

2008-01-01

Tmore » he possibility of using single-walled carbon nanotubes (SWCNs) aggregates as fluorescence sensors for pathogen recognition in drinking water treatment applications has been studied. Batch adsorption study is conducted to adsorb large concentrations of Staphylococcus aureus aureus SH 1000 and Escherichia coli pKV-11 on single-walled carbon nanotubes. Subsequently the immobilized bacteria are detected with confocal microscopy by coating the nanotubes with fluorescence emitting antibodies. he Freundlich adsorption equilibrium constant ( k ) for S.aureus and E.coli determined from batch adsorption study was found to be 9 × 10 8 and 2 × 10 8  ml/g, respectively. he visualization of bacterial cells adsorbed on fluorescently modified carbon nanotubes is also clearly seen. he results indicate that hydrophobic single-walled carbon nanotubes have excellent bacterial adsorption capacity and fluorescent detection capability. his is an important advancement in designing fluorescence biosensors for pathogen recognition in water systems.« less

Two-photon microscopy and spectroscopy based on a compact confocal scanning head

NASA Astrophysics Data System (ADS)

Diaspro, Alberto; Chirico, Giberto; Federici, Federico; Cannone, Fabio; Beretta, Sabrina; Robello, Mauro; Olivini, Francesca; Ramoino, Paola

2001-07-01

We have combined a confocal laser scanning head modified for TPE (two-photon excitation) microscopy with some spectroscopic modules to study single molecules and molecular aggregates. The behavior of the TPE microscope unit has been characterized by means of point spread function measurements and of the demonstration of its micropatterning abilities. One-photon and two-photon mode can be simply accomplished by switching from a mono-mode optical fiber (one-photon) coupled to conventional laser sources to an optical module that allows IR laser beam (two- photon/TPE) delivery to the confocal laser scanning head. We have then described the characterization of the two-photon microscope for spectroscopic applications: fluorescence correlation, lifetime and fluorescence polarization anisotropy measurements. We describe the measurement of the response of the two-photon microscope to the light polarization and discuss fluorescence polarization anisotropy measurements on Rhodamine 6G as a function of the viscosity and on a globular protein, the Beta-lactoglobulin B labeled with Alexa 532 at very high dilutions. The average rotational and translational diffusion coefficients measured with fluorescence polarization anisotropy and fluorescence correlation methods are in good agreement with the protein size, therefore validating the use of the microscope for two-photon spectroscopy on biomolecules.

Ultrasensitive investigations of biological systems by fluorescence correlation spectroscopy.

PubMed

Haustein, Elke; Schwille, Petra

2003-02-01

Fluorescence correlation spectroscopy (FCS) extracts information about molecular dynamics from the tiny fluctuations that can be observed in the emission of small ensembles of fluorescent molecules in thermodynamic equilibrium. Employing a confocal setup in conjunction with highly dilute samples, the average number of fluorescent particles simultaneously within the measurement volume (approximately 1 fl) is minimized. Among the multitude of chemical and physical parameters accessible by FCS are local concentrations, mobility coefficients, rate constants for association and dissociation processes, and even enzyme kinetics. As any reaction causing an alteration of the primary measurement parameters such as fluorescence brightness or mobility can be monitored, the application of this noninvasive method to unravel processes in living cells is straightforward. Due to the high spatial resolution of less than 0.5 microm, selective measurements in cellular compartments, e.g., to probe receptor-ligand interactions on cell membranes, are feasible. Moreover, the observation of local molecular dynamics provides access to environmental parameters such as local oxygen concentrations, pH, or viscosity. Thus, this versatile technique is of particular attractiveness for researchers striving for quantitative assessment of interactions and dynamics of small molecular quantities in biologically relevant systems.

Development of Fluorescence Surrogates to Predict the Photochemical Transformation of Pharmaceuticals in Wastewater Effluents.

PubMed

Yan, Shuwen; Yao, Bo; Lian, Lushi; Lu, Xinchen; Snyder, Shane A; Li, Rui; Song, Weihua

2017-03-07

The photochemical transformation of pharmaceutical and personal care products (PPCPs) in wastewater effluents is an emerging concern for environmental scientists. In the current study, the photodegradation of 29 PPCPs was examined in effluents under simulated solar irradiation. Direct photodegradation, triplet state effluent organic matter ( 3 EfOM*)-mediated and hydroxyl radical (HO • )-mediated degradation are three major pathways in the removal process. With the photodegradation of trace levels of PPCPs, the excitation-emission matrix (EEM) fluorescence intensities of the effluents were also gradually reduced. Therefore, fluorescence peaks have been identified, for the first time, as appropriate surrogates to assess the photodegradation of PPCPs. The humic-like fluorescence peak is linked to direct photolysis-labile PPCPs, such as naproxen, ronidazole, diclofenac, ornidazole, tinidazole, chloramphenicol, flumequine, ciprofloxacin, methadone, and dimetridazole. The tyrosine-like EEM peak is associated with HO • /CO 3 •- -labile PPCPs, such as trimethoprim, ibuprofen, gemfibrozil, atenolol, carbamazepine, and cephalexin. The tryptophan-like peak is associated with 3 EfOM*-labile PPCPs, such as clenbuterol, metoprolol, venlafaxine, bisphenol A, propranolol, ractopamine, salbutamol, roxithromycin, clarithromycin, azithromycin, famotidine, terbutaline, and erythromycin. The reduction in EEM fluorescence correlates well with the removal of PPCPs, allowing a model to be constructed. The solar-driven removal of EEM fluorescence was applied to predict the attenuation of 11 PPCPs in five field samples. A close correlation between the predicted results and the experimental results suggests that fluorescence may be a suitable surrogate for monitoring the solar-driven photodegradation of PPCPs in effluents.

Use of fluorescence EEM to monitor the removal of emerging contaminants in full scale wastewater treatment plants.

PubMed

Sgroi, Massimiliano; Roccaro, Paolo; Korshin, Gregory V; Greco, Valentina; Sciuto, Sebastiano; Anumol, Tarun; Snyder, Shane A; Vagliasindi, Federico G A

2017-02-05

This study investigated the applicability of different techniques for fluorescence excitation/emission matrices data interpretations, including peak-picking method, fluorescence regional integration and PARAFAC modelling, to act as surrogates in predicting emerging trace organic compounds (ETOrCs) removal during conventional wastewater treatments that usually comprise primary and secondary treatments. Results showed that fluorescence indexes developed using alternative methodologies but indicative of a same dissolved organic matter component resulted in similar predictions of the removal of the target compounds. The peak index defined by the excitation/emission wavelength positions (λ ex/ λ em ) 225/290nm and related to aromatic proteins and tyrosine-like fluorescence was determined to be a particularly suitable surrogate for monitoring ETOrCs that had very high removal rates (average removal >70%) (i.e., triclosan, caffeine and ibuprofen). The peak index defined by λ ex/ λ em =245/440nm and the PARAFAC component with wavelength of the maxima λ ex/ λ em =245, 350/450, both identified as humic-like fluorescence, were found remarkably well correlated with ETOrCs such as atenolol, naproxen and gemfibrozil that were moderately removed (51-70% average removal). Finally, the PARAFAC component with wavelength of the maxima λ ex/ λ em =<240, 315/380 identified as microbial humic-like fluorescence was the only index correlated with the removal of the antibiotic trimethoprim (average removal 68%). Copyright © 2016 Elsevier B.V. All rights reserved.

Speckle correlation resolution enhancement of wide-field fluorescence imaging (Conference Presentation)

NASA Astrophysics Data System (ADS)

Yilmaz, Hasan

2016-03-01

Structured illumination enables high-resolution fluorescence imaging of nanostructures [1]. We demonstrate a new high-resolution fluorescence imaging method that uses a scattering layer with a high-index substrate as a solid immersion lens [2]. Random scattering of coherent light enables a speckle pattern with a very fine structure that illuminates the fluorescent nanospheres on the back surface of the high-index substrate. The speckle pattern is raster-scanned over the fluorescent nanospheres using a speckle correlation effect known as the optical memory effect. A series of standard-resolution fluorescence images per each speckle pattern displacement are recorded by an electron-multiplying CCD camera using a commercial microscope objective. We have developed a new phase-retrieval algorithm to reconstruct a high-resolution, wide-field image from several standard-resolution wide-field images. We have introduced phase information of Fourier components of standard-resolution images as a new constraint in our algorithm which discards ambiguities therefore ensures convergence to a unique solution. We demonstrate two-dimensional fluorescence images of a collection of nanospheres with a deconvolved Abbe resolution of 116 nm and a field of view of 10 µm × 10 µm. Our method is robust against optical aberrations and stage drifts, therefore excellent for imaging nanostructures under ambient conditions. [1] M. G. L. Gustafsson, J. Microsc. 198, 82-87 (2000). [2] H. Yilmaz, E. G. van Putten, J. Bertolotti, A. Lagendijk, W. L. Vos, and A. P. Mosk, Optica 2, 424-429 (2015).

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.

Correlated FLIM and PLIM for cell metabolism

NASA Astrophysics Data System (ADS)

Rück, A.; Breymayer, J.; Kalinina, S.

2016-03-01

Correlated imaging of phosphorescence and fluorescence lifetime parameters of metabolic markers is a challenge for direct investigating mechanisms related to cell metabolism and oxygen tension. A large variety of clinical phenotypes is associated with mitochondrial defects accomplished with changes in cell metabolism. In many cases the hypoxic microenvironment of cancer cells shifts metabolism from oxidative phosphorylation (OXPHOS) to anaerobic or aerobic glycolysis, a process known as "Warburg" effect. Also during stem cell differentiation a switch in cell metabolism is observed. A defective mitochondrial function associated with hypoxia has been invoked in many complex disorders such as type 2 diabetes, Alzheimers disease, cardiac ischemia/reperfusion injury, tissue inflammation and cancer. Cellular responses to oxygen tension have been studied extensively, optical imaging techniques based on time correlated single photon counting (TCSPC) to detect the underlying metabolic mechanisms are therefore of prominent interest. They offer the possibility by inspecting fluorescence decay characteristics of intrinsic coenzymes to directly image metabolic pathways. Moreover oxygen tension can be determined by considering the phosphorescence lifetime of a phosphorescent probe. The combination of both fluorescence lifetime imaging (FLIM) of coenzymes like NADH and FAD and phosphorescence lifetime (PLIM) of phosphorescent dyes could provide valuable information about correlation of metabolic pathways and oxygen tension.

Quantitative Analysis of Self-Association and Mobility of Annexin A4 at the Plasma Membrane

PubMed Central

Crosby, Kevin C.; Postma, Marten; Hink, Mark A.; Zeelenberg, Christiaan H.C.; Adjobo-Hermans, Merel J.W.; Gadella, Theodorus W.J.

2013-01-01

Annexins, found in most eukaryotic species, are cytosolic proteins that are able to bind negatively-charged phospholipids in a calcium-dependent manner. Annexin A4 (AnxA4) has been implicated in diverse cellular processes, including the regulation of exocytosis and ion-transport; however, its precise mechanistic role is not fully understood. AnxA4 has been shown to aggregate on lipid layers upon Ca2+ binding in vitro, a characteristic that may be critical for its function. We have utilized advanced fluorescence microscopy to discern details on the mobility and self-assembly of AnxA4 after Ca2+ influx at the plasma membrane in living cells. Total internal reflection microscopy in combination with Förster resonance energy transfer reveals that there is a delay between initial plasma membrane binding and the beginning of self-assembly and this process continues after the cytoplasmic pool has completely relocated. Number-and-brightness analysis suggests that the predominant membrane bound mobile form of the protein is trimeric. There also exists a pool of AnxA4 that forms highly immobile aggregates at the membrane. Fluorescence recovery after photobleaching suggests that the relative proportion of these two forms varies and is correlated with membrane morphology. PMID:23663830

Quantitative analysis of self-association and mobility of annexin A4 at the plasma membrane.

PubMed

Crosby, Kevin C; Postma, Marten; Hink, Mark A; Zeelenberg, Christiaan H C; Adjobo-Hermans, Merel J W; Gadella, Theodorus W J

2013-05-07

Annexins, found in most eukaryotic species, are cytosolic proteins that are able to bind negatively-charged phospholipids in a calcium-dependent manner. Annexin A4 (AnxA4) has been implicated in diverse cellular processes, including the regulation of exocytosis and ion-transport; however, its precise mechanistic role is not fully understood. AnxA4 has been shown to aggregate on lipid layers upon Ca(2+) binding in vitro, a characteristic that may be critical for its function. We have utilized advanced fluorescence microscopy to discern details on the mobility and self-assembly of AnxA4 after Ca(2+) influx at the plasma membrane in living cells. Total internal reflection microscopy in combination with Förster resonance energy transfer reveals that there is a delay between initial plasma membrane binding and the beginning of self-assembly and this process continues after the cytoplasmic pool has completely relocated. Number-and-brightness analysis suggests that the predominant membrane bound mobile form of the protein is trimeric. There also exists a pool of AnxA4 that forms highly immobile aggregates at the membrane. Fluorescence recovery after photobleaching suggests that the relative proportion of these two forms varies and is correlated with membrane morphology. Copyright © 2013 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Confocal mosaicing microscopy of human skin ex vivo: spectral analysis for digital staining to simulate histology-like appearance

PubMed Central

Bini, Jason; Spain, James; Nehal, Kishwer; Hazelwood, Vikki; DiMarzio, Charles; Rajadhyaksha, Milind

2011-01-01

Confocal mosaicing microscopy enables rapid imaging of large areas of fresh tissue, without the processing that is necessary for conventional histology. Mosaicing may offer a means to perform rapid histology at the bedside. A possible barrier toward clinical acceptance is that the mosaics are based on a single mode of grayscale contrast and appear black and white, whereas histology is based on two stains (hematoxylin for nuclei, eosin for cellular cytoplasm and dermis) and appears purple and pink. Toward addressing this barrier, we report advances in digital staining: fluorescence mosaics that show only nuclei, are digitally stained purple and overlaid on reflectance mosaics, which show only cellular cytoplasm and dermis, and are digitally stained pink. With digital staining, the appearance of confocal mosaics mimics the appearance of histology. Using multispectral analysis and color matching functions, red, green, and blue (RGB) components of hematoxylin and eosin stains in tissue were determined. The resulting RGB components were then applied in a linear algorithm to transform fluorescence and reflectance contrast in confocal mosaics to the absorbance contrast seen in pathology. Optimization of staining with acridine orange showed improved quality of digitally stained mosaics, with good correlation to the corresponding histology. PMID:21806269

Biomarkers of aging in Drosophila.

PubMed

Jacobson, Jake; Lambert, Adrian J; Portero-Otín, Manuel; Pamplona, Reinald; Magwere, Tapiwanashe; Miwa, Satomi; Driege, Yasmine; Brand, Martin D; Partridge, Linda

2010-08-01

Low environmental temperature and dietary restriction (DR) extend lifespan in diverse organisms. In the fruit fly Drosophila, switching flies between temperatures alters the rate at which mortality subsequently increases with age but does not reverse mortality rate. In contrast, DR acts acutely to lower mortality risk; flies switched between control feeding and DR show a rapid reversal of mortality rate. Dietary restriction thus does not slow accumulation of aging-related damage. Molecular species that track the effects of temperatures on mortality but are unaltered with switches in diet are therefore potential biomarkers of aging-related damage. However, molecular species that switch upon instigation or withdrawal of DR are thus potential biomarkers of mechanisms underlying risk of mortality, but not of aging-related damage. Using this approach, we assessed several commonly used biomarkers of aging-related damage. Accumulation of fluorescent advanced glycation end products (AGEs) correlated strongly with mortality rate of flies at different temperatures but was independent of diet. Hence, fluorescent AGEs are biomarkers of aging-related damage in flies. In contrast, five oxidized and glycated protein adducts accumulated with age, but were reversible with both temperature and diet, and are therefore not markers either of acute risk of dying or of aging-related damage. Our approach provides a powerful method for identification of biomarkers of aging.

Biomarkers of ageing in Drosophila

PubMed Central

Jacobson, Jake; Portero-Otín, Manuel; Pamplona, Reinald; Magwere, Tapiwanashe; Miwa, Satomi; Driege, Yasmine; Brand, Martin D.; Partridge, Linda

2015-01-01

Summary Low environmental temperature and dietary restriction (DR) extend lifespan in diverse organisms. In the fruit fly Drosophila, switching flies between temperatures alters the rate at which mortality subsequently increases with age but does not reverse mortality rate. In contrast, DR acts acutely to lower mortality risk; flies switched between control feeding and DR show a rapid reversal of mortality rate. DR thus does not slow accumulation of ageing-related damage. Molecular species that track the effects of temperatures on mortality but are unaltered with switches in diet are therefore potential biomarkers of ageing-related damage. However, molecular species that switch upon instigation or withdrawal of DR are thus potential biomarkers of mechanisms underlying risk of mortality, but not of ageing-related damage. Using this approach, we assessed several commonly used biomarkers of ageing-related damage. Accumulation of fluorescent advanced glycation end products (AGEs) correlated strongly with mortality rate of flies at different temperatures but was independent of diet. Hence fluorescent AGEs are biomarkers of ageing-related damage in flies. In contrast, five oxidised and glycated protein adducts accumulated with age, but were reversible with both temperature and diet, and are therefore not markers either of acute risk of dying or of ageing-related damage. Our approach provides a powerful method for identification of biomarkers of ageing. PMID:20367621

Toward an integrative and predictive sperm quality analysis in Bos taurus.

PubMed

Yániz, J L; Soler, C; Alquézar-Baeta, C; Santolaria, P

2017-06-01

There is a need to develop more integrative sperm quality analysis methods, enabling researchers to evaluate different parameters simultaneously cell by cell. In this work, we present a new multi-parametric fluorescent test able to discriminate different sperm subpopulations based on their labeling pattern and motility characteristics. Cryopreserved semen samples from 20 Holstein bulls were used in the study. Analyses of sperm motility using computer-assisted sperm analysis (CASA-mot), membrane integrity by acridine orange-propidium iodide combination and multi-parametric by the ISAS ® 3Fun kit, were performed. The new method allows a clear discrimination of sperm subpopulations based on membrane and acrosomal integrity, motility and morphology. It was also possible to observe live spermatozoa showing signs of capacitation such as hyperactivated motility and changes in acrosomal structure. Sperm subpopulation with intact plasma membrane and acrosome showed a higher proportion of motile sperm than those with damaged acrosome or increased fluorescence intensity. Spermatozoa with intact plasmalemma and damaged acrosome were static or exhibit weak movement. Significant correlations among the different sperm quality parameters evaluated were also described. We concluded that the ISAS ® 3Fun is an integrated method that represents an advance in sperm quality analysis with the potential to improve fertility predictions. Copyright © 2017 Elsevier B.V. All rights reserved.

Development of fluorescent tracers for the real-time monitoring of renal function

NASA Astrophysics Data System (ADS)

Poreddy, Amruta R.; Asmelash, Bethel; Debreczeny, Martin P.; Fitch, Richard M.; Freskos, John N.; Galen, Karen P.; Gaston, Kimberly R.; Kostelc, James G.; Kumar, Rana; Marzan, Tim A.; Neumann, William L.; Rajagopalan, Raghavan; Schoenstein, Tasha M.; Shieh, Jeng-Jong; Wilcox, J. Micah; Wojdyla, Jolette K.; Dorshow, Richard B.

2011-03-01

Accurate measurement of glomerular filtration rate (GFR) at the bedside is highly desirable in order to assess renal function in real-time, which is currently an unmet clinical need. In our pursuit to develop exogenous fluorescent tracers as GFR markers, various hydrophilic derivatives of 3,6-diaminopyrazine-2,5-dicarboxylic acid with varying molecular weights and absorption/emission characteristics were synthesized. These include polyhydroxyalkyl based small molecules and poly(ethylene glycol) (PEG) substituted moderate molecular weight compounds, which were further sub-grouped into analogs having blue excitation with green emission, and relatively longer wavelength analogs having green excitation with orange emission. Lead compounds were identified in each of the four classes on the basis of structure- activity relationship studies, which included in vitro plasma protein binding, in vivo urine recovery of administered dose, and in vivo optical monitoring. The in vivo optical monitoring experiments with lead candidates have been correlated with plasma pharmacokinetic (PK) data for measurement of clearance and hence GFR. Renal clearance of these compounds, occurring exclusively via glomerular filtration, was established by probenecid blocking experiments. The renal clearance property of all these advanced candidates was superior to that of the iothalamate, which is currently an accepted standard for the measurement of GFR.

Tissue-specific effects of aldose reductase inhibition on fluorescence and cross-linking of extracellular matrix in chronic galactosemia. Relationship to pentosidine cross-links.

PubMed

Richard, S; Tamas, C; Sell, D R; Monnier, V M

1991-08-01

Chronic experimental hyperglycemia mediated by galactose has been shown to induce browning and cross-linking of rat tail tendon collagen that could be duplicated in vitro by nonenzymatic galactosylation. To investigate the nature of these changes, Sprague-Dawley rats were placed on a 33% galactose diet without and with sorbinil for 6 and 12 mo. Collagen-linked fluorescence and pentosidine cross-links increased with age and galactosemia in tail tendons (P less than 0.001) and skin but were essentially unresponsive to aldose reductase inhibition (ARI). In contrast, tendon breaking time in urea, a likely parameter of cross-linking, was markedly improved (P less than 0.001) by ARI. Fluorescence that was inhibited by sorbinil treatment was increased in pepsin and proteinase K digest of aortic tissue from galactosemic rats (P less than 0.001), but impaired enzymatic digestibility was not observed. Systolic blood pressure as potential consequence of aortic stiffening was not increased in galactosemia. These data suggest that fluorescence in skin and tendon might be in part due to advanced glycosylation and pentosidine formation because these were not decreased by ARI. However, they also suggest that nonfluorescent cross-links may also be forming because, in contrast to fluorescence, tail tendon breaking time was partly corrected by ARI. Thus, it appears that extracellular matrix changes in chronic galactosemia are complex, being partly attributable to advanced glycosylation and partly to polyol-pathway activation.

Applications of time-resolved laser fluorescence spectroscopy to the environmental biogeochemistry of actinides.

PubMed

Collins, Richard N; Saito, Takumi; Aoyagi, Noboru; Payne, Timothy E; Kimura, Takaumi; Waite, T David

2011-01-01

Time-resolved laser fluorescence spectroscopy (TRLFS) is a useful means of identifying certain actinide species resulting from various biogeochemical processes. In general, TRLFS differentiates chemical species of a fluorescent metal ion through analysis of different excitation and emission spectra and decay lifetimes. Although this spectroscopic technique has largely been applied to the analysis of actinide and lanthanide ions having fluorescence decay lifetimes on the order of microseconds, such as UO , Cm, and Eu, continuing development of ultra-fast and cryogenic TRLFS systems offers the possibility to obtain speciation information on metal ions having room-temperature fluorescence decay lifetimes on the order of nanoseconds to picoseconds. The main advantage of TRLFS over other advanced spectroscopic techniques is the ability to determine in situ metal speciation at environmentally relevant micromolar to picomolar concentrations. In the context of environmental biogeochemistry, TRLFS has principally been applied to studies of (i) metal speciation in aqueous and solid phases and (ii) the coordination environment of metal ions sorbed to mineral and bacterial surfaces. In this review, the principles of TRLFS are described, and the literature reporting the application of this methodology to the speciation of actinides in systems of biogeochemical interest is assessed. Significant developments in TRLFS methodology and advanced data analysis are highlighted, and we outline how these developments have the potential to further our mechanistic understanding of actinide biogeochemistry. American Society of Agronomy, Crop Science Society of America, and Soil Science Society of America.

Fluorescence spectroscopy of the retina from scrapie-infected mice

USDA-ARS?s Scientific Manuscript database

Recently, we have proposed that the fluorescence spectra of sheep retina can be well correlated to the presence or absence of scrapie. Scrapie is the most widespread TSE (transmissible spongiform encephalopathy) affecting sheep and goats worldwide. Mice eyes have been previously reported as a model ...

Recent Advances in Macrocyclic Fluorescent Probes for Ion Sensing.

PubMed

Wong, Joseph K-H; Todd, Matthew H; Rutledge, Peter J

2017-01-25

Small-molecule fluorescent probes play a myriad of important roles in chemical sensing. Many such systems incorporating a receptor component designed to recognise and bind a specific analyte, and a reporter or transducer component which signals the binding event with a change in fluorescence output have been developed. Fluorescent probes use a variety of mechanisms to transmit the binding event to the reporter unit, including photoinduced electron transfer (PET), charge transfer (CT), Förster resonance energy transfer (FRET), excimer formation, and aggregation induced emission (AIE) or aggregation caused quenching (ACQ). These systems respond to a wide array of potential analytes including protons, metal cations, anions, carbohydrates, and other biomolecules. This review surveys important new fluorescence-based probes for these and other analytes that have been reported over the past five years, focusing on the most widely exploited macrocyclic recognition components, those based on cyclam, calixarenes, cyclodextrins and crown ethers; other macrocyclic and non-macrocyclic receptors are also discussed.

High-Resolution “Fleezers”: Dual-Trap Optical Tweezers Combined with Single-Molecule Fluorescence Detection

PubMed Central

Whitley, Kevin D.; Comstock, Matthew J.; Chemla, Yann R.

2017-01-01

Recent advances in optical tweezers have greatly expanded their measurement capabilities. A new generation of hybrid instrument that combines nanomechanical manipulation with fluorescence detection—fluorescence optical tweezers, or “fleezers”—is providing a powerful approach to study complex macromolecular dynamics. Here, we describe a combined high-resolution optical trap/confocal fluorescence microscope that can simultaneously detect sub-nanometer displacements, sub-piconewton forces, and single-molecule fluorescence signals. The primary technical challenge to these hybrid instruments is how to combine both measurement modalities without sacrificing the sensitivity of either one. We present general design principles to overcome this challenge and provide detailed, step-by-step instructions to implement them in the construction and alignment of the instrument. Lastly, we present a set of protocols to perform a simple, proof-of-principle experiment that highlights the instrument capabilities. PMID:27844430

Optical Probes for Neurobiological Sensing and Imaging.

PubMed

Kim, Eric H; Chin, Gregory; Rong, Guoxin; Poskanzer, Kira E; Clark, Heather A

2018-05-15

Fluorescent nanosensors and molecular probes are next-generation tools for imaging chemical signaling inside and between cells. Electrophysiology has long been considered the gold standard in elucidating neural dynamics with high temporal resolution and precision, particularly on the single-cell level. However, electrode-based techniques face challenges in illuminating the specific chemicals involved in neural cell activation with adequate spatial information. Measuring chemical dynamics is of fundamental importance to better understand synergistic interactions between neurons as well as interactions between neurons and non-neuronal cells. Over the past decade, significant technological advances in optical probes and imaging methods have enabled entirely new possibilities for studying neural cells and circuits at the chemical level. These optical imaging modalities have shown promise for combining chemical, temporal, and spatial information. This potential makes them ideal candidates to unravel the complex neural interactions at multiple scales in the brain, which could be complemented by traditional electrophysiological methods to obtain a full spatiotemporal picture of neurochemical dynamics. Despite the potential, only a handful of probe candidates have been utilized to provide detailed chemical information in the brain. To date, most live imaging and chemical mapping studies rely on fluorescent molecular indicators to report intracellular calcium (Ca 2+ ) dynamics, which correlates with neuronal activity. Methodological advances for monitoring a full array of chemicals in the brain with improved spatial, temporal, and chemical resolution will thus enable mapping of neurochemical circuits with finer precision. On the basis of numerous studies in this exciting field, we review the current efforts to develop and apply a palette of optical probes and nanosensors for chemical sensing in the brain. There is a strong impetus to further develop technologies capable of probing entire neurobiological units with high spatiotemporal resolution. Thus, we introduce selected applications for ion and neurotransmitter detection to investigate both neurons and non-neuronal brain cells. We focus on families of optical probes because of their ability to sense a wide array of molecules and convey spatial information with minimal damage to tissue. We start with a discussion of currently available molecular probes, highlight recent advances in genetically modified fluorescent probes for ions and small molecules, and end with the latest research in nanosensors for biological imaging. Customizable, nanoscale optical sensors that accurately and dynamically monitor the local environment with high spatiotemporal resolution could lead to not only new insights into the function of all cell types but also a broader understanding of how diverse neural signaling systems act in conjunction with neighboring cells in a spatially relevant manner.

Advanced Imaging Approaches to Characterize Stromal and Metabolic Changes in In Vivo Mammary Tumor Models

DTIC Science & Technology

2013-03-01

characterization and toward future intravital studies. Preliminary fluorescence lifetime images were also collected intravitally through a mammary imaging window...intend to use this characterization to understand shifts in fluorescence lifetime collected by intravital imaging using a mammary imaging window...collected intravitally through a mammary imaging window implanted in a female, PyVT positive, Col1a1 heterozygote, mouse (Figure 7). A paper has

Maximizing the Biochemical Resolving Power of Fluorescence Microscopy

PubMed Central

Esposito, Alessandro; Popleteeva, Marina; Venkitaraman, Ashok R.

2013-01-01

Most recent advances in fluorescence microscopy have focused on achieving spatial resolutions below the diffraction limit. However, the inherent capability of fluorescence microscopy to non-invasively resolve different biochemical or physical environments in biological samples has not yet been formally described, because an adequate and general theoretical framework is lacking. Here, we develop a mathematical characterization of the biochemical resolution in fluorescence detection with Fisher information analysis. To improve the precision and the resolution of quantitative imaging methods, we demonstrate strategies for the optimization of fluorescence lifetime, fluorescence anisotropy and hyperspectral detection, as well as different multi-dimensional techniques. We describe optimized imaging protocols, provide optimization algorithms and describe precision and resolving power in biochemical imaging thanks to the analysis of the general properties of Fisher information in fluorescence detection. These strategies enable the optimal use of the information content available within the limited photon-budget typically available in fluorescence microscopy. This theoretical foundation leads to a generalized strategy for the optimization of multi-dimensional optical detection, and demonstrates how the parallel detection of all properties of fluorescence can maximize the biochemical resolving power of fluorescence microscopy, an approach we term Hyper Dimensional Imaging Microscopy (HDIM). Our work provides a theoretical framework for the description of the biochemical resolution in fluorescence microscopy, irrespective of spatial resolution, and for the development of a new class of microscopes that exploit multi-parametric detection systems. PMID:24204821

Fluorescent and Magnetic Mesoporous Hybrid Material: A Chemical and Biological Nanosensor for Hg2+ Ions

PubMed Central

Suresh, Moorthy; Anand, Chokkalingam; Frith, Jessica E.; Dhawale, Dattatray S.; Subramaniam, Vishnu P.; Strounina, Ekaterina; Sathish, Clastinrusselraj I.; Yamaura, Kazunari; Cooper-White, Justin J.; Vinu, Ajayan

2016-01-01

We introduce “sense, track and separate” approach for the removal of Hg2+ ion from aqueous media using highly ordered and magnetic mesoporous ferrosilicate nanocages functionalised with rhodamine fluorophore derivative. These functionalised materials offer both fluorescent and magnetic properties in a single system which help not only to selectively sense the Hg2+ ions with a high precision but also adsorb and separate a significant amount of Hg2+ ion in aqueous media. We demonstrate that the magnetic affinity of these materials, generated from the ultrafine γ-Fe2O3 nanoparticles present inside the nanochannels of the support, can efficiently be used as a fluorescent tag to sense the Hg2+ ions present in NIH3T3 fibroblasts live cells and to track the movement of the cells by external magnetic field monitored using confocal fluorescence microscopy. This simple approach of introducing multiple functions in the magnetic mesoporous materials raise the prospect of creating new advanced functional materials by fusing organic, inorganic and biomolecules to create advanced hybrid nanoporous materials which have a potential use not only for sensing and the separation of toxic metal ions but also for cell tracking in bio-separation and the drug delivery. PMID:26911660

Fluorescent photochromes of diarylethene series: synthesis and properties

NASA Astrophysics Data System (ADS)

Shirinian, Valerii Z.; Lonshakov, D. V.; Lvov, A. G.; Krayushkin, Mikhail M.

2013-06-01

The research data in the field of fluorescent photochromic di(het)arylethenes published over the last decade are summarized. The characteristics of these compounds significant for their application in the design of molecular optical memory systems and photocontrolled switches are considered. The main types of diarylethenes and methods for their synthesis are described, and the correlations between structure and spectral properties, in particular, fluorescence characteristics are analyzed. Considerable attention is given to the means for endowing diarylethenes with fluorescence properties as one of the most promising methods for data readout from molecular information carriers. The bibliography includes 203 references.

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.

Understanding photon sideband statistics and correlation for determining phonon coherence

NASA Astrophysics Data System (ADS)

Ding, Ding; Yin, Xiaobo; Li, Baowen

2018-01-01

Generating and detecting coherent high-frequency heat-carrying phonons have been topics of great interest in recent years. Although there have been successful attempts in generating and observing coherent phonons, rigorous techniques to characterize and detect phonon coherence in a crystalline material have been lagging compared to what has been achieved for photons. One main challenge is a lack of detailed understanding of how detection signals for phonons can be related to coherence. The quantum theory of photoelectric detection has greatly advanced the ability to characterize photon coherence in the past century, and a similar theory for phonon detection is necessary. Here, we reexamine the optical sideband fluorescence technique that has been used to detect high-frequency phonons in materials with optically active defects. We propose a quantum theory of phonon detection using the sideband technique and found that there are distinct differences in sideband counting statistics between thermal and coherent phonons. We further propose a second-order correlation function unique to sideband signals that allows for a rigorous distinction between thermal and coherent phonons. Our theory is relevant to a correlation measurement with nontrivial response functions at the quantum level and can potentially bridge the gap of experimentally determining phonon coherence to be on par with that of photons.

Objective automated quantification of fluorescence signal in histological sections of rat lens.

PubMed

Talebizadeh, Nooshin; Hagström, Nanna Zhou; Yu, Zhaohua; Kronschläger, Martin; Söderberg, Per; Wählby, Carolina

2017-08-01

Visual quantification and classification of fluorescent signals is the gold standard in microscopy. The purpose of this study was to develop an automated method to delineate cells and to quantify expression of fluorescent signal of biomarkers in each nucleus and cytoplasm of lens epithelial cells in a histological section. A region of interest representing the lens epithelium was manually demarcated in each input image. Thereafter, individual cell nuclei within the region of interest were automatically delineated based on watershed segmentation and thresholding with an algorithm developed in Matlab™. Fluorescence signal was quantified within nuclei, cytoplasms and juxtaposed backgrounds. The classification of cells as labelled or not labelled was based on comparison of the fluorescence signal within cells with local background. The classification rule was thereafter optimized as compared with visual classification of a limited dataset. The performance of the automated classification was evaluated by asking 11 independent blinded observers to classify all cells (n = 395) in one lens image. Time consumed by the automatic algorithm and visual classification of cells was recorded. On an average, 77% of the cells were correctly classified as compared with the majority vote of the visual observers. The average agreement among visual observers was 83%. However, variation among visual observers was high, and agreement between two visual observers was as low as 71% in the worst case. Automated classification was on average 10 times faster than visual scoring. The presented method enables objective and fast detection of lens epithelial cells and quantification of expression of fluorescent signal with an accuracy comparable with the variability among visual observers. © 2017 International Society for Advancement of Cytometry. © 2017 International Society for Advancement of Cytometry.

Construction of a 'turn-on' fluorescent probe system for His-tagged proteins.

PubMed

Murata, Atsushi; Arai, Satoshi; Yoon, Su-In; Takabayashi, Masao; Ozaki, Miwako; Takeoka, Shinji

2010-12-01

Hexahistidine ((His)(6)) tags are used to purify genetically engineered proteins. Herein, we describe the construction of a 'turn-on' fluorescent probe system that consists of the fluorescence quencher, Dabcyl, conjugated to (His)(6), and fluorescent tetramethylrhodamine conjugated to nitrilotriacetic acid, which, in the presence of Ni(2+), can bind (His)(6). The system is turned off when Dabcyl-(His)(6) is bound to the fluorescent nitrilotriacetic acid derivative. The binding strength of this system was assessed using electrospray ionization mass spectrometry, fluorescence correlation spectroscopy, and fluorescence intensity distribution analysis-polarization. Although there was no significant enhancement in fluorescence after addition of an equimolar amount of ubiquitin, the fluorescence increased from 14% to 40% of its initial intensity when an equimolar amount of (His)(6)-ubiquitin was added. Therefore, this system should be able to specifically recognize (His)(6)-proteins with good resolution and has the additional advantage that a washing step is not required to remove fluorescent probe, that is, not bound to the (His)(6)-protein. Copyright © 2010 Elsevier Ltd. All rights reserved.

Determination of biological activity from fluorescence-lifetime measurements in Saccharomyces cerevisiae

NASA Astrophysics Data System (ADS)

Rudek, F.; Baselt, T.; Lempe, B.; Taudt, C.; Hartmann, P.

2015-03-01

The importance of fluorescence lifetime measurement as an optical analysis tool is growing. Many applications already exist in order to determine the fluorescence lifetime, but the majority of these require the addition of fluorescence-active substances to enable measurements. Every usage of such foreign materials has an associated risk. This paper investigates the use of auto-fluorescing substances in Saccharomyces cerevisiae (Baker's yeast) as a risk free alternative to fluorescence-active substance enabled measurements. The experimental setup uses a nitrogen laser with a pulse length of 350 ps and a wavelength of 337 nm. The excited sample emits light due to fluorescence of NADH/NADPH and collagen. A fast photodiode collects the light at the output of an appropriate high-pass edge-filter at 400 nm. Fluorescence lifetimes can be determined from the decay of the measurement signals, which in turn characterizes the individual materials and their surrounding environment. Information about the quantity of the fluorescence active substances can also be measured based on the received signal intensity. The correlation between the fluorescence lifetime and the metabolic state of Saccharomyces cerevisiae was investigated and is presented here.

Fluorescence, Absorption, and Excitation Spectra of Polycyclic Aromatic Hydrocarbons as a Tool for Quantitative Analysis

ERIC Educational Resources Information Center

Rivera-Figueroa, A. M.; Ramazan, K. A.; Finlayson-Pitts, B. J.

2004-01-01

A quantitative and qualitative study of the interplay between absorption, fluorescence, and excitation spectra of pollutants called polycyclic aromatic hydrocarbons (PAHs) is conducted. The study of five PAH displays the correlation of the above-mentioned properties along with the associated molecular changes.

Fluorescence hyperspectral imaging technique for the foreign substance detection on fresh-cut lettuce

USDA-ARS?s Scientific Manuscript database

Nondestructive methods based on fluorescence hyperspectral imaging (HSI) techniques were developed in order to detect worms on fresh-cut lettuce. The optimal wavebands for detecting worms on fresh-cut lettuce were investigated using the one-way ANOVA analysis and correlation analysis. The worm detec...

Accumulation of carboxymethyl-lysine (CML) in human cortical bone.

PubMed

Thomas, Corinne J; Cleland, Timothy P; Sroga, Grazyna E; Vashishth, Deepak

2018-05-01

Advanced glycation end-products (AGEs) are a category of post translational modification associated with the degradation of the structural properties of multiple different types of tissues. Typically, AGEs are the result of a series of post-translational modification reactions between sugars and proteins through a process known as non-enzymatic glycation (NEG). Increases in the rate of NEG of bone tissue are associated with type 2 diabetes and skeletal fragility. Current methods of assessing NEG and its impact on bone fracture risk involve measurement of pentosidine or total fluorescent AGEs (fAGEs). However, pentosidine represents only a small fraction of possible fAGEs present in bone, and neither pentosidine nor total fAGE measurement accounts for non-fluorescent AGEs, which are known to form in significant amounts in skin and other collagenous tissues. Carboxymethyl-lysine (CML) is a non-fluorescent AGE that is often measured and has been shown to accumulate in tissues such as skin, heart, arteries, and intervertebral disks, but is currently not assessed in bone. Here we show the localization of CML to collagen I using mass spectrometry for the first time in human bone. We then present a new method using demineralization followed by heating and trypsin digestion to measure CML content in human bone and demonstrate that CML in bone is 40-100 times greater than pentosidine (the current most commonly used marker of AGEs in bone). We then establish the viability of CML as a measurable AGE in bone by showing that levels of CML, obtained from bone using this technique, increase with age (p<0.05) and are correlated with previously reported measures of bone toughness. Thus, CML is a viable non-fluorescent AGE target to assess AGE accumulation and fragility in bone. The method developed here to extract and measure CML from human bone could facilitate the development of a new diagnostic assay to evaluate fracture risk and potentially lead to new therapeutic approaches to address bone fragility. Copyright © 2018 Elsevier Inc. All rights reserved.

Optical fluorescence spectroscopy to detect hepatic necrosis after normothermic ischemia: animal model

NASA Astrophysics Data System (ADS)

Romano, Renan A.; Vollet-Filho, Jose D.; Pratavieira, Sebastião.; Fernandez, Jorge L.; Kurachi, Cristina; Bagnato, Vanderlei S.; Castro-e-Silva, Orlando; Sankarankutty, Ajith K.

2015-06-01

Liver transplantation is a well-established treatment for liver failure. However, the success of the transplantation procedure depends on liver graft conditions. The tissue function evaluation during the several transplantation stages is relevant, in particular during the organ harvesting, when a decision is made concerning the viability of the graft. Optical fluorescence spectroscopy is a good option because it is a noninvasive and fast technique. A partial normothermic hepatic ischemia was performed in rat livers, with a vascular occlusion of both median and left lateral lobes, allowing circulation only for the right lateral lobe and the caudate lobe. Fluorescence spectra under excitation at 532 nm (doubled frequency Nd:YAG laser) were collected using a portable spectrometer (USB2000, Ocean Optics, USA). The fluorescence emission was collected before vascular occlusion, after ischemia, and 24 hours after reperfusion. A morphometric histology analysis was performed as the gold standard evaluation - liver samples were analyzed, and the percentage of necrotic tissue was obtained. The results showed that changes in the fluorescence emission after ischemia can be correlated with the amount of necrosis evaluated by a morphometric analysis, the Pearson correlation coefficient of the generated model was 0.90 and the root mean square error was around 20%. In this context, the laser-induced fluorescence spectroscopy technique after normothermic ischemia showed to be a fast and efficient method to differentiate ischemic injury from viable tissues.

In vivo quantification of chromophore concentration using fluorescence differential path length spectroscopy

NASA Astrophysics Data System (ADS)

Kruijt, Bastiaan; Kascakova, Slavka; de Bruijn, Henriette S.; van der Ploeg-van den Heuvel, Angelique; Sterenborg, Henricus J. C. M.; Robinson, Dominic J.; Amelink, Arjen

2009-05-01

We present an optical method based on fluorescence spectroscopy for measuring chromophore concentrations in vivo. Fluorescence differential path length spectroscopy (FPDS) determines chromophore concentration based on the fluorescence intensity corrected for absorption. The concentration of the photosensitizer m-THPC (Foscan®) was studied in vivo in normal rat liver, which is highly vascularized and therefore highly absorbing. Concentration estimates of m-THPC measured by FDPS on the liver are compared with chemical extraction. Twenty-five rats were injected with 0.3 mg/kg m-THPC. In vivo optical concentration measurements were performed on tissue 3, 24, 48, and 96 h after m-THPC administration to yield a 10-fold variation in tissue concentration. After the optical measurements, the liver was harvested for chemical extraction. FDPS showed good correlation with chemical extraction. FDPS also showed a correlation between m-THPC fluorescence and blood volume fraction at the two shortest drug-light intervals. This suggests different compartmental localization of m-THPC for different drug-light intervals that can be resolved using fluorescence spectroscopy. Differences in measured m-THPC concentration between FDPS and chemical extraction are related to the interrogation volume of each technique; ~0.2 mm3 and ~102 mm3, respectively. This indicates intra-animal variation in m-THPC distribution in the liver on the scale of the FDPS sampling volume.

Noninvasive measurement of pharmacokinetics by near-infrared fluorescence imaging in the eye of mice

NASA Astrophysics Data System (ADS)

Dobosz, Michael; Strobel, Steffen; Stubenrauch, Kay-Gunnar; Osl, Franz; Scheuer, Werner

2014-01-01

Purpose: For generating preclinical pharmacokinetics (PKs) of compounds, blood is drawn at different time points and levels are quantified by different analytical methods. In order to receive statistically meaningful data, 3 to 5 animals are used for each time point to get serum peak-level and half-life of the compound. Both characteristics are determined by data interpolation, which may influence the accuracy of these values. We provide a method that allows continuous monitoring of blood levels noninvasively by measuring the fluorescence intensity of labeled compounds in the eye and other body regions of anesthetized mice. Procedures: The method evaluation was performed with four different fluorescent compounds: (i) indocyanine green, a nontargeting dye; (ii) OsteoSense750, a bone targeting agent; (iii) tumor targeting Trastuzumab-Alexa750; and (iv) its F(-alxea750 fragment. The latter was used for a direct comparison between fluorescence imaging and classical blood analysis using enzyme-linked immunosorbent assay (ELISA). Results: We found an excellent correlation between blood levels measured by noninvasive eye imaging with the results generated by classical methods. A strong correlation between eye imaging and ELISA was demonstrated for the F( fragment. Whole body imaging revealed a compound accumulation in the expected regions (e.g., liver, bone). Conclusions: The combination of eye and whole body fluorescence imaging enables the simultaneous measurement of blood PKs and biodistribution of fluorescent-labeled compounds.

Note: a 4 ns hardware photon correlator based on a general-purpose field-programmable gate array development board implemented in a compact setup for fluorescence correlation spectroscopy.

PubMed

Kalinin, Stanislav; Kühnemuth, Ralf; Vardanyan, Hayk; Seidel, Claus A M

2012-09-01

We present a fast hardware photon correlator implemented in a field-programmable gate array (FPGA) combined with a compact confocal fluorescence setup. The correlator has two independent units with a time resolution of 4 ns while utilizing less than 15% of a low-end FPGA. The device directly accepts transistor-transistor logic (TTL) signals from two photon counting detectors and calculates two auto- or cross-correlation curves in real time. Test measurements demonstrate that the performance of our correlator is comparable with the current generation of commercial devices. The sensitivity of the optical setup is identical or even superior to current commercial devices. The FPGA design and the optical setup both allow for a straightforward extension to multi-color applications. This inexpensive and compact solution with a very good performance can serve as a versatile platform for uses in education, applied sciences, and basic research.

Note: A 4 ns hardware photon correlator based on a general-purpose field-programmable gate array development board implemented in a compact setup for fluorescence correlation spectroscopy

NASA Astrophysics Data System (ADS)

Kalinin, Stanislav; Kühnemuth, Ralf; Vardanyan, Hayk; Seidel, Claus A. M.

2012-09-01

We present a fast hardware photon correlator implemented in a field-programmable gate array (FPGA) combined with a compact confocal fluorescence setup. The correlator has two independent units with a time resolution of 4 ns while utilizing less than 15% of a low-end FPGA. The device directly accepts transistor-transistor logic (TTL) signals from two photon counting detectors and calculates two auto- or cross-correlation curves in real time. Test measurements demonstrate that the performance of our correlator is comparable with the current generation of commercial devices. The sensitivity of the optical setup is identical or even superior to current commercial devices. The FPGA design and the optical setup both allow for a straightforward extension to multi-color applications. This inexpensive and compact solution with a very good performance can serve as a versatile platform for uses in education, applied sciences, and basic research.

Noninvasive detection of diabetes mellitus

NASA Astrophysics Data System (ADS)

Eppstein, Jonathan A.; Bursell, Sven-Erik

1992-05-01

Recent advances in fluorescence spectroscopy of the lens reveal the potential of a non-invasive device and methodology to sensitively measure changes in the lens of the eye associated with diabetes mellitus. The system relies on the detection of the spectrum of fluorescence emitted from a selected volume (approximately 1/10 mm3) of the lens of living human subjects using low power excitation illumination from monochromatic light sources. The sensitivity of this technique is based on the measurement of the fluorescence intensity in a selected region of the fluorescence spectrum and normalization of this fluorescence with respect to attenuation (scattering and absorption) of the incident excitation light. The amplitude of the unshifted Rayleigh line, measured as part of the fluorescence spectrum, is used as a measure of the attenuation of the excitation light in the lens. Using this methodology we have demonstrated that the normalized lens fluorescence provides a more sensitive discrimination between diabetic and non-diabetic lenses than more conventional measurements of fluorescence intensity from the lens. The existing instrumentation will be described as well as the proposed design for a commercial version of the instrument expected to be ready for FDA trials by late 1992. The results from clinical measurements are used to describe a relationship between normalized lens fluorescence and hemoglobin A1c levels in diabetic patients.

Fluorescent genetic barcoding in mammalian cells for enhanced multiplexing capabilities in flow cytometry.

PubMed

Smurthwaite, Cameron A; Hilton, Brett J; O'Hanlon, Ryan; Stolp, Zachary D; Hancock, Bryan M; Abbadessa, Darin; Stotland, Aleksandr; Sklar, Larry A; Wolkowicz, Roland

2014-01-01

The discovery of the green fluorescent protein from Aequorea victoria has revolutionized the field of cell and molecular biology. Since its discovery a growing panel of fluorescent proteins, fluorophores and fluorescent-coupled staining methodologies, have expanded the analytical capabilities of flow cytometry. Here, we exploit the power of genetic engineering to barcode individual cells with genes encoding fluorescent proteins. For genetic engineering, we utilize retroviral technology, which allows for the expression of ectopic genetic information in a stable manner in mammalian cells. We have genetically barcoded both adherent and nonadherent cells with different fluorescent proteins. Multiplexing power was increased by combining both the number of distinct fluorescent proteins, and the fluorescence intensity in each channel. Moreover, retroviral expression has proven to be stable for at least a 6-month period, which is critical for applications such as biological screens. We have shown the applicability of fluorescent barcoded multiplexing to cell-based assays that rely themselves on genetic barcoding, or on classical staining protocols. Fluorescent genetic barcoding gives the cell an inherited characteristic that distinguishes it from its counterpart. Once cell lines are developed, no further manipulation or staining is required, decreasing time, nonspecific background associated with staining protocols, and cost. The increasing number of discovered and/or engineered fluorescent proteins with unique absorbance/emission spectra, combined with the growing number of detection devices and lasers, increases multiplexing versatility, making fluorescent genetic barcoding a powerful tool for flow cytometry-based analysis. © 2013 International Society for Advancement of Cytometry.

In vitro performance of DIAGNOdent laser fluorescence device for dental calculus detection on human tooth root surfaces.

PubMed

Rams, Thomas E; Alwaqyan, Abdulaziz Y

2017-10-01

This study assessed the reproducibility of a red diode laser device, and its capability to detect dental calculus in vitro on human tooth root surfaces. On each of 50 extracted teeth, a calculus-positive and calculus-free root surface was evaluated by two independent examiners with a low-power indium gallium arsenide phosphide diode laser (DIAGNOdent) fitted with a periodontal probe-like sapphire tip and emitting visible red light at 655 nm wavelength. Laser autofluorescence intensity readings of examined root surfaces were scored on a 0-99 scale, with duplicate assessments performed using the laser probe tip directed both perpendicular and parallel to evaluated tooth root surfaces. Pearson correlation coefficients of untransformed measurements, and kappa analysis of data dichotomized with a >40 autofluorescence intensity threshold, were calculated to assess intra- and inter-examiner reproducibility of the laser device. Mean autofluorescence intensity scores of calculus-positive and calculus-free root surfaces were evaluated with the Student's t -test. Excellent intra- and inter-examiner reproducibility was found for DIAGNOdent laser autofluorescence intensity measurements, with Pearson correlation coefficients above 94%, and kappa values ranging between 0.96 and 1.0, for duplicate readings taken with both laser probe tip orientations. Significantly higher autofluorescence intensity values were measured when the laser probe tip was directed perpendicular, rather than parallel, to tooth root surfaces. However, calculus-positive roots, particularly with calculus in markedly-raised ledges, yielded significantly greater mean DIAGNOdent laser autofluorescence intensity scores than calculus-free surfaces, regardless of probe tip orientation. DIAGNOdent autofluorescence intensity values >40 exhibited a stronger association with calculus (36.6 odds ratio) then measurements of ≥5 (20.1 odds ratio) when the laser probe tip was advanced parallel to root surfaces. Excellent intra- and inter-examiner reproducibility of autofluorescence intensity measurements was obtained with the DIAGNOdent laser fluorescence device on human tooth roots. Calculus-positive root surfaces exhibited significantly greater DIAGNOdent laser autofluorescence than calculus-free tooth roots, even with the laser probe tip directed parallel to root surfaces. These findings provide further in vitro validation of the potential utility of a DIAGNOdent laser fluorescence device for identifying dental calculus on human tooth root surfaces.

Stochastic Simulation of Dopamine Neuromodulation for Implementation of Fluorescent Neurochemical Probes in the Striatal Extracellular Space.

PubMed

Beyene, Abraham G; McFarlane, Ian R; Pinals, Rebecca L; Landry, Markita P

2017-10-18

Imaging the dynamic behavior of neuromodulatory neurotransmitters in the extracelluar space that arise from individual quantal release events would constitute a major advance in neurochemical imaging. Spatial and temporal resolution of these highly stochastic neuromodulatory events requires concurrent advances in the chemical development of optical nanosensors selective for neuromodulators in concert with advances in imaging methodologies to capture millisecond neurotransmitter release. Herein, we develop and implement a stochastic model to describe dopamine dynamics in the extracellular space (ECS) of the brain dorsal striatum to guide the design and implementation of fluorescent neurochemical probes that record neurotransmitter dynamics in the ECS. Our model is developed from first-principles and simulates release, diffusion, and reuptake of dopamine in a 3D simulation volume of striatal tissue. We find that in vivo imaging of neuromodulation requires simultaneous optimization of dopamine nanosensor reversibility and sensitivity: dopamine imaging in the striatum or nucleus accumbens requires nanosensors with an optimal dopamine dissociation constant (K d ) of 1 μM, whereas K d s above 10 μM are required for dopamine imaging in the prefrontal cortex. Furthermore, as a result of the probabilistic nature of dopamine terminal activity in the striatum, our model reveals that imaging frame rates of 20 Hz are optimal for recording temporally resolved dopamine release events. Our work provides a modeling platform to probe how complex neuromodulatory processes can be studied with fluorescent nanosensors and enables direct evaluation of nanosensor chemistry and imaging hardware parameters. Our stochastic model is generic for evaluating fluorescent neurotransmission probes, and is broadly applicable to the design of other neurotransmitter fluorophores and their optimization for implementation in vivo.

Stream Dissolved Organic Matter Quantity and Quality Along a Wetland-Cropland Catchment Gradient

NASA Astrophysics Data System (ADS)

McDonough, O.; Hosen, J. D.; Lang, M. W.; Oesterling, R.; Palmer, M.

2012-12-01

Wetlands may be critical sources of dissolved organic matter (DOM) to stream networks. Yet, more than half of wetlands in the continental United States have been lost since European settlement, with the majority of loss attributed to agriculture. The degree to which agricultural loss of wetlands impacts stream DOM is largely unknown and may have important ecological implications. Using twenty headwater catchments on the Delmarva Peninsula (Maryland, USA), we investigated the seasonal influence of wetland and cropland coverage on downstream DOM quantity and quality. In addition to quantifying bulk downstream dissolved organic carbon (DOC) concentration, we used a suite of DOM UV-absorbance metrics and parallel factor analysis (PARAFAC) modeling of excitation-emission fluorescence spectra (EEMs) to characterize DOM composition. Percent bioavailable DOC (%BDOC) was measured during the Spring sampling using a 28-day incubation. Percent wetland coverage and % cropland within the watersheds were significantly negatively correlated (r = -0.93, p < 0.001). Results show that % wetland coverage was positively correlated with stream DOM concentration, molecular weight, aromaticity, humic-like fluorescence, and allochthonous origin. Conversely, increased wetland coverage was negatively correlated with stream DOM protein-like fluorescence. Percent BDOC decreased with DOM humic-like fluorescence and increased with protein-like fluorescence. We observed minimal seasonal interaction between % wetland coverage and DOM concentration and composition across Spring, Fall, and Winter sampling seasons. However, principal component analysis suggested more pronounced seasonal differences exist in stream DOM. This study highlights the influence of wetlands on downstream DOM in agriculturally impacted landscapes where loss of wetlands to cultivation may significantly alter stream DOM quantity and quality.

Fluorescence microscopy for the characterization of structural integrity

NASA Technical Reports Server (NTRS)

Street, Kenneth W.; Leonhardt, Todd A.

1991-01-01

The absorption characteristics of light and the optical technique of fluorescence microscopy for enhancing metallographic interpretation are presented. Characterization of thermally sprayed coatings by optical microscopy suffers because of the tendency for misidentification of the microstructure produced by metallographic preparation. Gray scale, in bright field microscopy, is frequently the only means of differentiating the actual structural details of porosity, cracking, and debonding of coatings. Fluorescence microscopy is a technique that helps to distinguish the artifacts of metallographic preparation (pullout, cracking, debonding) from the microstructure of the specimen by color contrasting structural differences. Alternative instrumentation and the use of other dye systems are also discussed. The combination of epoxy vacuum infiltration with fluorescence microscopy to verify microstructural defects is an effective means to characterize advanced materials and to assess structural integrity.

Complementary Spectroscopic Assays for Investigating Protein-Ligand Binding Activity: A Project for the Advanced Chemistry Laboratory

ERIC Educational Resources Information Center

Mascotti, David P.; Waner, Mark J.

2010-01-01

A protein-ligand binding, guided-inquiry laboratory project with potential application across the advanced undergraduate curriculum is described. At the heart of the project are fluorescence and spectrophotometric assays utilizing biotin-4-fluorescein and streptavidin. The use of the same stock solutions for an assay that may be examined by two…

Evaluation of inhibition of cancer cell proliferation in vitro with different berries and correlation with their antioxidant levels by advanced analytical methods.

PubMed

Flis, Sylwia; Jastrzebski, Zenon; Namiesnik, Jacek; Arancibia-Avila, Patricia; Toledo, Fernando; Leontowicz, Hanna; Leontowicz, Maria; Suhaj, Milan; Trakhtenberg, Simon; Gorinstein, Shela

2012-03-25

Dimethylsulfoxide extracts of Chilean berries [Myrteola nummularia, 'Murtilla-like'] vs. well known 'Murtilla', Chilean and Polish blueberries, and Chilean raspberries were investigated for their antioxidant, quenching and antiproliferative activities. The significantly highest levels of polyphenols, flavonoids, flavanols and their antioxidant activities were estimated in 'Murtilla' (MT) berries (P<0.05), than in other investigated samples. DPPH kinetic measurements were calculated to compare, distinguish and discriminate the antiradical activity among berry extracts by multivariate analysis. The lowest IC(50) values, 751 and 858 μg/ml, were obtained for MT extract on colon cancer cell lines HT-29 and SW48. HT-29 cells treated with MT extract showed a decrease in G1 phase cells from 77% to 56% (P<0.05). At the highest concentration of 2000 μg/ml MT extract caused 90-100% cell growth inhibition. Percentage of death cells treated with MT extract was 80.1% and 72.5% for SW48 and HT-29 cells, respectively. The inhibition of cancer cell proliferation highly correlated with the levels of polyphenols, flavonoids and their antioxidant activities. The interaction between drugs and serum albumin plays an important role in the distribution and metabolism of drugs, therefore the complexation reaction between flavonoids, and berries extracts, and bovine serum albumin (BSA) was investigated by 3-D fluorescence and FTIR spectroscopy. The results indicated that flavonoids and polyphenol extracts have strong ability to quench the intrinsic fluorescence of BSA by forming complexes. A shift in the maximum of amides FTIR-bands appeared. In conclusion, these findings suggest that the intake of a new kind of berry, as a source of natural antioxidants, may reduce colon cancer risk. Copyright © 2012 Elsevier B.V. All rights reserved.

Revealing the Effects of Nanoscale Membrane Curvature on Lipid Mobility

PubMed Central

Kabbani, Abir Maarouf; Woodward, Xinxin

2017-01-01

Recent advances in nanoengineering and super-resolution microscopy have enabled new capabilities for creating and observing membrane curvature. However, the effects of curvature on single-lipid diffusion have yet to be revealed. The simulations presented here describe the capabilities of varying experimental methods for revealing the effects of nanoscale curvature on single-molecule mobility. Traditionally, lipid mobility is revealed through fluorescence recovery after photobleaching (FRAP), fluorescence correlation spectroscopy (FCS), and single particle tracking (SPT). However, these techniques vary greatly in their ability to detect the effects of nanoscale curvature on lipid behavior. Traditionally, FRAP and FCS depend on diffraction-limited illumination and detection. A simulation of FRAP shows minimal effects on lipids diffusion due to a 50 nm radius membrane bud. Throughout the stages of the budding process, FRAP detected minimal changes in lipid recovery time due to the curvature versus flat membrane. Simulated FCS demonstrated small effects due to a 50 nm radius membrane bud that was more apparent with curvature-dependent lipid mobility changes. However, SPT achieves a sub-diffraction-limited resolution of membrane budding and lipid mobility through the identification of the single-lipid positions with ≤15 nm spatial and ≤20 ms temporal resolution. By mapping the single-lipid step lengths to locations on the membrane, the effects of membrane topography and curvature could be correlated to the effective membrane viscosity. Single-fluorophore localization techniques, such SPT, can detect membrane curvature and its effects on lipid behavior. These simulations and discussion provide a guideline for optimizing the experimental procedures in revealing the effects of curvature on lipid mobility and effective local membrane viscosity. PMID:29057801

Non-Constant Learning Rates in Retrospective Experience Curve Analyses and their Correlation to Deployment Programs

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

Wei, Max; Smith, Sarah J.; Sohn, Michael D.

2015-07-16

A key challenge for policy-makers and technology market forecasters is to estimate future technology costs and in particular the rate of cost reduction versus production volume. A related, critical question is what role should state and federal governments have in advancing energy efficient and renewable energy technologies? This work provides retrospective experience curves and learning rates for several energy-related technologies, each of which have a known history of federal and state deployment programs. We derive learning rates for eight technologies including energy efficient lighting technologies, stationary fuel cell systems, and residential solar photovoltaics, and provide an overview and timeline ofmore » historical deployment programs such as state and federal standards and state and national incentive programs for each technology. Piecewise linear regimes are observed in a range of technology experience curves, and public investments or deployment programs are found to be strongly correlated to an increase in learning rate across multiple technologies. A downward bend in the experience curve is found in 5 out of the 8 energy-related technologies presented here (electronic ballasts, magnetic ballasts, compact fluorescent lighting, general service fluorescent lighting, and the installed cost of solar PV). In each of the five downward-bending experience curves, we believe that an increase in the learning rate can be linked to deployment programs to some degree. This work sheds light on the endogenous versus exogenous contributions to technological innovation and highlights the impact of exogenous government sponsored deployment programs. This work can inform future policy investment direction and can shed light on market transformation and technology learning behavior.« less

Determination of ethambutol by a sensitive fluorescent probe

NASA Astrophysics Data System (ADS)

Wu, Wen-Ying; Yang, Ji-Yuan; Du, Li-Ming; Wu, Hao; Li, Chang-Feng

2011-08-01

The competitive reaction between ethambutol and two fluorescent probes (i.e., berberine and palmatine) for occupancy of the cucurbit[7]uril (CB[7]) cavity was studied by spectrofluorometry. The CB[7] reacts with these probes to form stable complexes, and the fluorescence intensity of the complexes is greatly enhanced. In addition, the excitation and emission wavelengths of their complexes moved to wavelengths of 343 nm and 495 nm, respectively. However, the addition of ethambutol dramatically quenches the fluorescence intensity of the two complexes. Accordingly, a couple of new fluorescence quenching methods for the determination of ethambutol were established. The methods can be applied for quantifying ethambutol. A linear relationship between the fluorescence quenching values (Δ F) and ethambutol concentration exists in the range of 5.0-1000.0 ng mL -1, with a correlation coefficient ( r) of 0.9997. The detection limit is 1.7 ng mL -1. The fluorescent probe of berberine has higher sensitivity than palmatine. This paper also discusses the mechanism of fluorescence indicator probes.

Correlative Förster Resonance Electron Transfer-Proximity Ligation Assay (FRET-PLA) Technique for Studying Interactions Involving Membrane Proteins.

PubMed

Ivanusic, Daniel; Denner, Joachim; Bannert, Norbert

2016-08-01

This unit provides a guide and detailed protocol for studying membrane protein-protein interactions (PPI) using the acceptor-sensitized Förster resonance electron transfer (FRET) method in combination with the proximity ligation assay (PLA). The protocol in this unit is focused on the preparation of FRET-PLA samples and the detection of correlative FRET/PLA signals as well as on the analysis of FRET-PLA data and interpretation of correlative results when using cyan fluorescent protein (CFP) as a FRET donor and yellow fluorescent protein (YFP) as a FRET acceptor. The correlative application of FRET and PLA combines two powerful tools for monitoring PPI, yielding results that are more reliable than with either technique alone. © 2016 by John Wiley & Sons, Inc. Copyright © 2016 John Wiley & Sons, Inc.

Correlations of trace elements in breast human tissues: Evaluation of spatial distribution using μ-XRF

NASA Astrophysics Data System (ADS)

Silva, Marina Piacenti da; Silva, Deisy Mara da; Ribeiro-Silva, Alfredo; Poletti, Martin Eduardo

2012-05-01

The aim of this work is to investigate microscopic correlations between trace elements in breast human tissues. A synchrotron X-ray fluorescence microprobe system (μ-XRF) was used to obtain two-dimensional distribution of trace element Ca, Fe, Cu and Zn in normal (6 samples) and malignant (14 samples) breast tissues. The experiment was performed in X-ray Fluorescence beam line at Laboratório Nacional de Luz Síncrotron (LNLS), Campinas, Brazil. The white microbeam was generated with a fine conical capillary with a 20 μm output diameter. The samples were supported on a XYZ table. An optical microscope with motorized zoom was used for sample positioning and choice the area to be scanned. Automatic two-dimensional scans were programmed and performed with steps of 30 μm in each direction (x, y) on the selected area. The fluorescence signals were recorded using a Si(Li) detector, positioned at 90 degrees with respect to the incident beam, with a collection time of 10 s per point. The elemental maps obtained from each sample were overlap to observe correlation between trace elements. Qualitative results showed that the pairs of elements Ca-Zn and Fe-Cu could to be correlated in malignant breast tissues. Quantitative results, achieved by Spearman correlation tests, indicate that there is a spatial correlation between these pairs of elements (p < 0.001) suggesting the importance of these elements in metabolic processes associated with the development of the tumor.

Online fluorescence spectroscopy for the real-time evaluation of the microbial quality of drinking water.

PubMed

Sorensen, J P R; Vivanco, A; Ascott, M J; Gooddy, D C; Lapworth, D J; Read, D S; Rushworth, C M; Bucknall, J; Herbert, K; Karapanos, I; Gumm, L P; Taylor, R G

2018-06-15

We assessed the utility of online fluorescence spectroscopy for the real-time evaluation of the microbial quality of untreated drinking water. Online fluorimeters were installed on the raw water intake at four groundwater-derived UK public water supplies alongside existing turbidity sensors that are used to forewarn of the presence of microbial contamination in the water industry. The fluorimeters targeted fluorescent dissolved organic matter (DOM) peaks at excitation/emission wavelengths of 280/365 nm (tryptophan-like fluorescence, TLF) and 280/450 nm (humic-like fluorescence, HLF). Discrete samples were collected for Escherichia coli, total bacterial cell counts by flow cytometry, and laboratory-based fluorescence and absorbance. Both TLF and HLF were strongly correlated with E. coli (ρ = 0.71-0.77) and total bacterial cell concentrations (ρ = 0.73-0.76), whereas the correlations between turbidity and E. coli (ρ = 0.48) and total bacterial cell counts (ρ = 0.40) were much weaker. No clear TLF peak was observed at the sites and all apparent TLF was considered to be optical bleed-through from the neighbouring HLF peak. Therefore, a HLF fluorimeter alone would be sufficient to evaluate the microbial water quality at these sources. Fluorescent DOM was also influenced by site operations such as pump start-up and the precipitation of cations on the sensor windows. Online fluorescent DOM sensors are a better indicator of the microbial quality of untreated drinking water than turbidity and they have wide-ranging potential applications within the water industry. Copyright © 2018 British Geological Survey, a component institute of NERC - 'BGS © NERC 2018'. Published by Elsevier Ltd.. All rights reserved.

Estimating chlorophyll content and photochemical yield of photosystem II (ΦPSII) using solar-induced chlorophyll fluorescence measurements at different growing stages of attached leaves

PubMed Central

Tubuxin, Bayaer; Rahimzadeh-Bajgiran, Parinaz; Ginnan, Yusaku; Hosoi, Fumiki; Omasa, Kenji

2015-01-01

This paper illustrates the possibility of measuring chlorophyll (Chl) content and Chl fluorescence parameters by the solar-induced Chl fluorescence (SIF) method using the Fraunhofer line depth (FLD) principle, and compares the results with the standard measurement methods. A high-spectral resolution HR2000+ and an ordinary USB4000 spectrometer were used to measure leaf reflectance under solar and artificial light, respectively, to estimate Chl fluorescence. Using leaves of Capsicum annuum cv. ‘Sven’ (paprika), the relationships between the Chl content and the steady-state Chl fluorescence near oxygen absorption bands of O2B (686nm) and O2A (760nm), measured under artificial and solar light at different growing stages of leaves, were evaluated. The Chl fluorescence yields of ΦF 686nm/ΦF 760nm ratios obtained from both methods correlated well with the Chl content (steady-state solar light: R2 = 0.73; artificial light: R2 = 0.94). The SIF method was less accurate for Chl content estimation when Chl content was high. The steady-state solar-induced Chl fluorescence yield ratio correlated very well with the artificial-light-induced one (R2 = 0.84). A new methodology is then presented to estimate photochemical yield of photosystem II (ΦPSII) from the SIF measurements, which was verified against the standard Chl fluorescence measurement method (pulse-amplitude modulated method). The high coefficient of determination (R2 = 0.74) between the ΦPSII of the two methods shows that photosynthesis process parameters can be successfully estimated using the presented methodology. PMID:26071530

A fluorescent probe-labeled Escherichia coli aspartate transcarbamoylase that monitors the allosteric conformational state.

PubMed

West, Jay M; Tsuruta, Hiro; Kantrowitz, Evan R

2004-01-09

A new system has been developed capable of monitoring conformational changes of the 240s loop of aspartate transcarbamoylase, which are tightly correlated with the quaternary structural transition, with high sensitivity in solution. Pyrene, a fluorescent probe, was conjugated to residue 241 in the 240s loop of aspartate transcarbamoylase to monitor changes in conformation by fluorescence spectroscopy. Pyrene maleimide was conjugated to a cysteine residue on the 240s loop of a previously constructed double catalytic chain mutant version of the enzyme, C47A/A241C. The pyrene-labeled enzyme undergoes the normal T to R structural transition, as demonstrated by small-angle x-ray scattering. Like the wild-type enzyme, the pyrene-labeled enzyme exhibits cooperativity toward aspartate, and is activated by ATP and inhibited by CTP at subsaturating concentrations of aspartate. The binding of the bisubstrate analogue N-(phosphonoacetyl)-l-aspartate (PALA), or the aspartate analogue succinate, in the presence of saturating carbamoyl phosphate, to the pyrenelabeled enzyme caused a sigmoidal change in the fluorescence emission. Saturation with ATP and CTP (in the presence of either subsaturating amounts of PALA or succinate and carbamoyl phosphate) caused a hyperbolic increase and decrease, respectively, in the fluorescence emission. The half-saturation values from the fluorescence saturation curves and kinetic saturation curves were, within error, identical. Fluorescence and small-angle x-ray scattering stopped-flow experiments, using aspartate and carbamoyl phosphate, confirm that the change in excimer fluorescence and the quaternary structure change correlate. These results in conjunction with previous studies suggest that the allosteric transition involves both global and local conformational changes and that the heterotropic effect of the nucleotides may be exerted through local conformational changes in the active site by directly influencing the conformation of the 240s loop.

Frequency-domain phase fluorometry in the presence of dark states: A numerical study

NASA Astrophysics Data System (ADS)

Zhu, Xinxin; Min, Wei

2011-11-01

Fluorescence anomalous phase advance (FAPA) is a newly discovered spectroscopy phenomenon: instead of lagging behind the modulated light, fluorescence signal can exhibit FAPA as if it precedes the excitation source in time. While FAPA offers a promising technique for probing dark state lifetime, the underlying mechanism is not fully elucidated. Herein we investigate frequency-domain phase fluorometry as a result of intricate interplay between a short-lived fluorescent state and a long-lived dark state. In particular, the quantitative dependence on modulation frequency, excitation intensity, nonradiative decay, intersystem crossing and dark-state lifetime are explored respectively. A comprehensive view of phase fluorometry emerges consequently.

Fluorescence-guided tumor visualization using a custom designed NIR attachment to a surgical microscope for high sensitivity imaging (Conference Presentation)

NASA Astrophysics Data System (ADS)

Kittle, David S.; Patil, Chirag G.; Mamelak, Adam; Hansen, Stacey; Perry, Jeff; Ishak, Laura; Black, Keith L.; Butte, Pramod V.

2016-03-01

Current surgical microscopes are limited in sensitivity for NIR fluorescence. Recent developments in tumor markers attached with NIR dyes require newer, more sensitive imaging systems with high resolution to guide surgical resection. We report on a small, single camera solution enabling advanced image processing opportunities previously unavailable for ultra-high sensitivity imaging of these agents. The system captures both visible reflectance and NIR fluorescence at 300 fps while displaying full HD resolution video at 60 fps. The camera head has been designed to easily mount onto the Zeiss Pentero microscope head for seamless integration into surgical procedures.

Microwave-induced facile synthesis of water-soluble fluorogenic alginic acid derivatives.

PubMed

Chhatbar, Mahesh U; Meena, Ramavatar; Prasad, Kamalesh; Chejara, Dharmesh R; Siddhanta, A K

2011-04-01

A facile microwave-induced method was developed for synthesizing water-soluble fluorescent derivatives of alginic acid (ALG) with four different diamines, hydrazine (HY), ethylenediamine (EDA), 1,6-hexanediamine (HDA), and 1,4-cyclohexanediamine (CHDA), followed by a cross-linking reaction with a natural cross linker genipin. The ethylenediamine derivative of alginic acid (ALG-EDA) exhibited good fluorescent activity, which upon cross linking was enhanced threefold. The other amide derivatives, for example, ALG-HY, ALG-HDA, and ALG-CHDA, were not fluorescent, but their respective crosslinked products exhibited excellent fluorescent activity. The fluorescence intensity had an inverse correlation with the number of carbon atoms present in the amine, which in turn was a function of degree of substitution (DS). These fluorescent polysaccharide derivatives are of potential utility in the domain of sensor applications. Copyright © 2011 Elsevier Ltd. All rights reserved.

Vectorized data acquisition and fast triple-correlation integrals for Fluorescence Triple Correlation Spectroscopy

NASA Astrophysics Data System (ADS)

Ridgeway, William K.; Millar, David P.; Williamson, James R.

2013-04-01

Fluorescence Correlation Spectroscopy (FCS) is widely used to quantify reaction rates and concentrations of molecules in vitro and in vivo. We recently reported Fluorescence Triple Correlation Spectroscopy (F3CS), which correlates three signals together instead of two. F3CS can analyze the stoichiometries of complex mixtures and detect irreversible processes by identifying time-reversal asymmetries. Here we report the computational developments that were required for the realization of F3CS and present the results as the Triple Correlation Toolbox suite of programs. Triple Correlation Toolbox is a complete data analysis pipeline capable of acquiring, correlating and fitting large data sets. Each segment of the pipeline handles error estimates for accurate error-weighted global fitting. Data acquisition was accelerated with a combination of off-the-shelf counter-timer chips and vectorized operations on 128-bit registers. This allows desktop computers with inexpensive data acquisition cards to acquire hours of multiple-channel data with sub-microsecond time resolution. Off-line correlation integrals were implemented as a two delay time multiple-tau scheme that scales efficiently with multiple processors and provides an unprecedented view of linked dynamics. Global fitting routines are provided to fit FCS and F3CS data to models containing up to ten species. Triple Correlation Toolbox is a complete package that enables F3CS to be performed on existing microscopes. Catalogue identifier: AEOP_v1_0 Program summary URL:http://cpc.cs.qub.ac.uk/summaries/AEOP_v1_0.html Program obtainable from: CPC Program Library, Queen’s University, Belfast, N. Ireland Licensing provisions: Standard CPC licence, http://cpc.cs.qub.ac.uk/licence/licence.html No. of lines in distributed program, including test data, etc.: 50189 No. of bytes in distributed program, including test data, etc.: 6135283 Distribution format: tar.gz Programming language: C/Assembly. Computer: Any with GCC and library support. Operating system: Linux and OS X (data acq. for Linux only due to library availability), not tested on Windows. RAM: ≥512 MB. Classification: 16.4. External routines: NIDAQmx (National Instruments), Gnu Scientific Library, GTK+, PLplot (optional) Nature of problem: Fluorescence Triple Correlation Spectroscopy required three things: data acquisition at faster speeds than were possible without expensive custom hardware, triple-correlation routines that could process 1/2 TB data sets rapidly, and fitting routines capable of handling several to a hundred fit parameters and 14,000 + data points, each with error estimates. Solution method: A novel data acquisition concept mixed signal processing with off-the-shelf hardware and data-parallel processing using 128-bit registers found in desktop CPUs. Correlation algorithms used fractal data structures and multithreading to reduce data analysis times. Global fitting was implemented with robust minimization routines and provides feedback that allows the user to critically inspect initial guesses and fits. Restrictions: Data acquisition only requires a National Instruments data acquisition card (it was tested on Linux using card PCIe-6251) and a simple home-built circuit. Unusual features: Hand-coded ×86-64 assembly for data acquisition loops (platform-independent C code also provided). Additional comments: A complete collection of tools to perform Fluorescence Triple Correlation Spectroscopy-from data acquisition to two-tau correlation of large data sets, to model fitting. Running time: 1-5 h of data analysis per hour of data collected. Varies depending on data-acquisition length, time resolution, data density and number of cores used for correlation integrals.

Assessing delivery and quantifying efficacy of small interfering ribonucleic acid therapeutics in the skin using a dual-axis confocal microscope

NASA Astrophysics Data System (ADS)

Ra, Hyejun; Gonzalez-Gonzalez, Emilio; Smith, Bryan R.; Gambhir, Sanjiv S.; Kino, Gordon S.; Solgaard, Olav; Kaspar, Roger L.; Contag, Christopher H.

2010-05-01

Transgenic reporter mice and advances in imaging instrumentation are enabling real-time visualization of cellular mechanisms in living subjects and accelerating the development of novel therapies. Innovative confocal microscope designs are improving their utility for microscopic imaging of fluorescent reporters in living animals. We develop dual-axis confocal (DAC) microscopes for such in vivo studies and create mouse models where fluorescent proteins are expressed in the skin for the purpose of advancing skin therapeutics and transdermal delivery tools. Three-dimensional image volumes, through the different skin compartments of the epidermis and dermis, can be acquired in several seconds with the DAC microscope in living mice, and are comparable to histologic analyses of reporter protein expression patterns in skin sections. Intravital imaging with the DAC microscope further enables visualization of green fluorescent protein (GFP) reporter gene expression in the skin over time, and quantification of transdermal delivery of small interfering RNA (siRNA) and therapeutic efficacy. Visualization of transdermal delivery of nucleic acids will play an important role in the development of innovative strategies for treating skin pathologies.

The use of near-infrared fluorescence imaging in endocrine surgical procedures.

PubMed

Kahramangil, Bora; Berber, Eren

2017-06-01

Near-infrared fluorescence imaging in endocrine surgery is a new, yet highly investigated area. It involves indocyanine green use as well as parathyroid autofluorescence. Several groups have described their technique and reported on the observed utility. However, there is no consensus on technical details. Furthermore, the correlation between intraoperative findings and postoperative outcomes is unclear. With this study, we aim to review the current literature on fluorescence imaging and share our insights on technical details. © 2017 Wiley Periodicals, Inc.

The use of serology by titering of fluorescent antibodies to evaluate levels of transmission of schistosomiasis in Rhodesia.

PubMed

Shiff, C J; Yiannakis, C

1976-05-01

The prevalence of schistosomiasis has been measured by parasitological examination and by fluorescent antibody titering in a number of communities in different parts of Rhodesia. The samples were taken from areas of high, medium, and low transmission of the disease. A correlation was detected between the mean titer and prevalence of infection, particularly in the younger people (4-13 years old). It is suggested that fluorescent antibody titrating may be a useful epidemiological tool.

Spectroscopic Evidence of Anthropogenic Compounds Extraction from Polymers by Fluorescent Dissolved Organic Matter in Natural Water

NASA Astrophysics Data System (ADS)

Miranda, M.; Trojzuck, A.; Voss, D.; Gassmann, S.; Zielinski, O.

2016-04-01

FDOM is one of the most important carriers of anthropogenic compounds in natural waters. It can combine with environmental contaminants and polymers to form diverse chemical structures. To this end, here a microfluidic chip was designed for the analysis of these changes in fluorescent dissolved organic matter (FDOM) fingerprints due to thermal treatment and varying time intervals of exposure. Excitation Emission Matrix Spectroscopy (EEMS) approach was utilized to detect and identify the inherent compounds in sampled FDOM. Strong direct correlations were founded, Spearman rank correlation values (ρ = 0.85 at α = 0.1, n = 4) and linear correlation R2 = 0.8359 were noted between thermal treatment pattern 2 and fluorescence intensity of samples. Materials, acrylic based glue and cyclic olefin copolymer (COC) polymer, used to design the microfluidic sensor were determined to possess unique spectral features in the ultraviolet to green spectrum using EEMS. The study therefore provides an insight on methods to identify contaminants in natural waters. This underlines the potential of optical sensors providing measurements at fast intervals, enabling environmental monitoring.

Multiple emitters in a waveguide: Nonreciprocity and correlated photons at perfect elastic transmission

NASA Astrophysics Data System (ADS)

Fang, Yao-Lung L.; Baranger, Harold U.

2017-07-01

We investigate interference and correlation effects when several detuned emitters are placed along a one-dimensional photonic waveguide. Such a setup allows multiple interactions between the photons and the strongly coupled emitters, and underlies proposed devices for quantum information processing. We show, first, that a pair of detuned two-level systems (2LS) separated by a half wavelength mimic a driven Λ -type three-level system (3LS) in both the single- and two-photon sectors. There is an interference-induced transparency peak at which the fluorescence is quenched, leaving the transmitted photons completely uncorrelated. Slightly away from this separation, we find that the inelastic scattering (fluorescence) is large, leading to nonlinear effects such as nonreciprocity (rectification). We connect this nonreciprocity to inelastic scattering caused by driving a dark pole and so derive a condition for maximum rectification. Finally, by placing a true 3LS midway between the two 2LS, we show that elastic scattering produces only transmission, but inelastic scattering nevertheless occurs (the fluorescence is not quenched) causing substantial photon correlations.

Probing transcription factor diffusion dynamics in the living mammalian embryo with photoactivatable fluorescence correlation spectroscopy.

PubMed

Kaur, Gurpreet; Costa, Mauro W; Nefzger, Christian M; Silva, Juan; Fierro-González, Juan Carlos; Polo, Jose M; Bell, Toby D M; Plachta, Nicolas

2013-01-01

Transcription factors use diffusion to search the DNA, yet the mechanisms controlling transcription factor diffusion during mammalian development remain poorly understood. Here we combine photoactivation and fluorescence correlation spectroscopy to study transcription factor diffusion in developing mouse embryos. We show that the pluripotency-associated transcription factor Oct4 displays both fast and Brownian and slower subdiffusive behaviours that are controlled by DNA interactions. Following cell lineage specification, the slower DNA-interacting diffusion fraction distinguishes pluripotent from extraembryonic cell nuclei. Similar to Oct4, Sox2 shows slower diffusion in pluripotent cells while Cdx2 displays opposite dynamics, suggesting that slow diffusion may represent a general feature of transcription factors in lineages where they are essential. Slow Oct4 subdiffusive behaviours are conserved in embryonic stem cells and induced pluripotent stem cells (iPS cells), and lost during differentiation. We also show that Oct4 diffusion depends on its interaction with ERG-associated protein with SET domain. Photoactivation and fluorescence correlation spectroscopy provides a new intravital approach to study transcription factor diffusion in complex in vivo systems.

Precise correlation of histopathological and fluorescein angiographic morphology using retinal vascular casting.

PubMed

Bek, T; Prause, J U

1996-12-01

The histopathology of three eyes obtained post mortem from 2 patients with age-related macular degeneration was correlated with the pre mortem fluorescein angiographic morphology. A precise point-by-point correlation between histopathology and the corresponding angiographic appearance was ensured by using the cast retinal vascular system as a pattern of reference. The study showed that both the photoreceptors, the pigment epithelium, and substances accumulated between the retinal and the choroidal vascular systems, may have a blocking effect on choroidal background fluorescence as seen on fluorescein angiograms. Furthermore, it is confirmed that fluorescein angiographic hyperfluorescence may be due to a lack of blocking of the choroidal fluorescence because of a window defect in the retinal photoreceptor layer and/or the pigment epithelium.

Laser diagnostics of an evaporating electrospray

NASA Astrophysics Data System (ADS)

Yi, Tongxun

2014-01-01

An electrospray atomizer generates monodisperse, dilute sprays when working in the cone-jet mode. Evolution of an electrospray with droplet diameter below 10 μm is studied with phase Doppler particle analyzer (PDPA) and the exciplex-PLIF technique. The evaporation rate constant is determined from droplet velocity and diameter measured with a PDPA and is found to sharply increase with the velocity slip and the coflow temperature. Fluorescence around 400 nm, usually referred to as TMPD fluorescence, is calibrated with a heated, laminar, coflow vapor jet diluted with nitrogen. The TMPD fluorescence yield nonlinearly increases with temperature up to 538 K and then declines. Single-shot images show that fluorescence around 400 nm is mainly generated from TMPD vapor and that from droplets can be neglected as a first analysis; however, fluorescence around 490 nm, usually referred to as exciplex fluorescence, is generated from both droplets and fuel vapor immediately around droplets. Exciplex fluorescence is correlated with PDPA measurements and TMPD fluorescence. Effects of temperature, fuel composition, overlap of fluorescent spectra, and chemical equilibrium for exciplex formation are discussed. Technical challenges for quantitative exciplex-PLIF measurements are highlighted.

Relationship between the Fluorescence Lifetime of Chlorophyll 'a' and Primary Productivity within the Mississippi River Plume and Adjacent Shelf Region

NASA Technical Reports Server (NTRS)

Hall, Callie; Miller, Richard L.; Fernandez, Salvador M.; McKee, Brent A.

2000-01-01

In situ measurements of chlorophyll fluorescence intensity have been widely used to estimate phytoplankton biomass. However, because the fluorescence quantum yield of chlorophyll a in vivo can be highly variable, measurements of chlorophyll fluorescence intensity cannot be directly correlated with phytoplankton biomass and do not provide information on the physiological state of the phytoplankton under study. Conversely, lifetime-based measurements of chlorophyll fluorescence provide a framework in which photosynthetic rates of phytoplankton can be analyzed according to phytoplankton physiology. Along with the measurement of primary production and ambient nutrient concentrations within the Mississippi River plume in the northern Gulf of Mexico, phytoplankton fluorescence lifetimes were measured using a Fluorescence Lifetime Phytoplankton Analyzer (developed under a NASA Small Business Innovative Research contract to Ciencia, Inc.). Variability of fluorescence lifetimes within the plume can be used as a background from which to interpret variations in the maximum quantum yield of photochemistry. The extent to which nutrient and effluent loading in this dynamic coastal area affect the photosynthetic performance of phytoplankton will be presented as a function of phytoplankton fluorescence lifetimes.

Fluorescence spectroscopy for the detection of potentially malignant disorders of the oral cavity: analysis of 30 cases

NASA Astrophysics Data System (ADS)

Francisco, A. L. N.; Correr, W. R.; Azevedo, L. H.; Galletta, V. K.; Pinto, C. A. L.; Kowalski, L. P.; Kurachi, C.

2014-01-01

Oral cancer is a major health problem worldwide and although early diagnosis of potentially malignant and malignant diseases is associated with better treatment results, a large number of cancers are initially misdiagnosed, with unfortunate consequences for long-term survival. Fluorescence spectroscopy is a noninvasive modality of diagnostic approach using induced fluorescence emission in tumors that can improve diagnostic accuracy. The objective of this study was to determine the ability to discriminate between normal oral mucosa and potentially malignant disorders by fluorescence spectroscopy. Fluorescence investigation under 408 and 532 nm excitation wavelengths was performed on 60 subjects, 30 with potentially malignant disorders and 30 volunteers with normal mucosa. Data was analyzed to correlate fluorescence patterns with clinical and histopathological diagnostics. Fluorescence spectroscopy used as a point measurement technique resulted in a great variety of spectral information. In a qualitative analysis of the fluorescence spectral characteristics of each type of injury evaluated, it was possible to discriminate between normal and abnormal oral mucosa. The results show the potential use of fluorescence spectroscopy for an improved discrimination of oral disorders.

Reliable measurement of E. coli single cell fluorescence distribution using a standard microscope set-up.

PubMed

Cortesi, Marilisa; Bandiera, Lucia; Pasini, Alice; Bevilacqua, Alessandro; Gherardi, Alessandro; Furini, Simone; Giordano, Emanuele

2017-01-01

Quantifying gene expression at single cell level is fundamental for the complete characterization of synthetic gene circuits, due to the significant impact of noise and inter-cellular variability on the system's functionality. Commercial set-ups that allow the acquisition of fluorescent signal at single cell level (flow cytometers or quantitative microscopes) are expensive apparatuses that are hardly affordable by small laboratories. A protocol that makes a standard optical microscope able to acquire quantitative, single cell, fluorescent data from a bacterial population transformed with synthetic gene circuitry is presented. Single cell fluorescence values, acquired with a microscope set-up and processed with custom-made software, are compared with results that were obtained with a flow cytometer in a bacterial population transformed with the same gene circuitry. The high correlation between data from the two experimental set-ups, with a correlation coefficient computed over the tested dynamic range > 0.99, proves that a standard optical microscope- when coupled with appropriate software for image processing- might be used for quantitative single-cell fluorescence measurements. The calibration of the set-up, together with its validation, is described. The experimental protocol described in this paper makes quantitative measurement of single cell fluorescence accessible to laboratories equipped with standard optical microscope set-ups. Our method allows for an affordable measurement/quantification of intercellular variability, whose better understanding of this phenomenon will improve our comprehension of cellular behaviors and the design of synthetic gene circuits. All the required software is freely available to the synthetic biology community (MUSIQ Microscope flUorescence SIngle cell Quantification).

Seasonal variability in CDOM absorption and fluorescence properties in the Barataria Basin, Louisiana, USA.

PubMed

Singh, Shatrughan; D'Sa, Eurico; Swenson, Erick

2010-01-01

Absorption and fluorescence properties of chromophoric dissolved organic matter (CDOM) along a 124 km transect in the Barataria Basin, a large estuary located in Louisiana, USA, were investigated during high and low flow periods of the Mississippi River in the spring and winter of 2008-2009. Mean CDOM absorption at 355 nm from the marine to the freshwater end member stations ranged from (3.25 +/- 0.56) to (20.76 +/- 2.43) m(-1) for the three month high flow period whereas it varied from (1.48 +/- 1.08) to (25.45 +/- 7.03) m(-1) for the same stations during low flow period. Corresponding salinity values at these stations indicated the influence of river and shelf exchanges in the lower basin and precipitation and runoff in the upper basin. An inverse relationship of CDOM absorbance and fluorescence with salinity observed in the basin could be a useful indicator of salinity. CDOM fluorescence also varied over a large range showing an approximately 8 to 12-fold increase between the marine and freshwater end members for the two flow seasons. Excitation-emission matrix spectral plots indicated the presence of various fluorescence components with highest being the A-peak, lowest the T-peak, and the C and M-peaks showing similar trends along the transect. During low flow season the A/C ratio were well correlated with station locations indicating increased terrestrial influence towards the upper basin. CDOM absorption and fluorescence at 355 nm were highly correlated and independent of CDOM sources suggesting that fluorescence could be used to characterize CDOM in the basin.

Factors Affecting the Quantification of Biomolecular Interactions by Fluorescence Cross-Correlation Spectroscopy

PubMed Central

Foo, Yong Hwee; Naredi-Rainer, Nikolaus; Lamb, Don C.; Ahmed, Sohail; Wohland, Thorsten

2012-01-01

Fluorescence cross-correlation spectroscopy (FCCS) is used to determine interactions and dissociation constants (Kds) of biomolecules. The determination of a Kd depends on the accurate measurement of the auto- and cross-correlation function (ACF and CCF) amplitudes. In the case of complete binding, the ratio of the CCF/ACF amplitudes is expected to be 1. However, measurements performed on tandem fluorescent proteins (FPs), in which two different FPs are linked, yield CCF/ACF amplitude ratios of ∼0.5 or less for different FCCS schemes. We use single wavelength FCCS and pulsed interleaved excitation FCCS to measure various tandem FPs constituted of different red and green FPs and determine the causes for this suboptimal ratio. The main causes for the reduced CCF/ACF amplitude ratio are differences in observation volumes for the different labels, the existence of dark FPs due to maturation problems, photobleaching, and to a lesser extent Förster (or fluorescence) resonance energy transfer between the labels. We deduce the fraction of nonfluorescent proteins for EGFP, mRFP, and mCherry as well as the differences in observation volumes. We use this information to correct FCCS measurements of the interaction of Cdc42, a small Rho-GTPase, with its effector IQGAP1 in live cell measurements to obtain a label-independent value for the Kd. PMID:22404940

Near-infrared fluorescence imaging of experimentally collagen-induced arthritis in rats using the nonspecific dye tetrasulfocyanine in comparison with gadolinium-based contrast-enhanced magnetic resonance imaging, histology, and clinical score

NASA Astrophysics Data System (ADS)

Gemeinhardt, Ines; Puls, Dorothee; Gemeinhardt, Ole; Taupitz, Matthias; Wagner, Susanne; Schnorr, Beatrix; Licha, Kai; Schirner, Michael; Ebert, Bernd; Petzelt, Diethard; Macdonald, Rainer; Schnorr, Jörg

2012-10-01

Using 15 rats with collagen-induced arthritis (30 joints) and 7 control rats (14 joints), we correlated the intensity of near-infrared fluorescence (NIRF) of the nonspecific dye tetrasulfocyanine (TSC) with magnetic resonance imaging (MRI), histopathology, and clinical score. Fluorescence images were obtained in reflection geometry using a NIRF camera system. Normalized fluorescence intensity (INF) was determined after intravenous dye administration on different time points up to 120 min. Contrast-enhanced MRI using gadodiamide was performed after NIRF imaging. Analyses were performed in a blinded fashion. Histopathological and clinical scores were determined for each ankle joint. INF of moderate and high-grade arthritic joints were significantly higher (p<0.005) than the values of control and low-grade arthritic joints between 5 and 30 min after TSC-injection. This result correlated well with post-contrast MRI signal intensities at about 5 min after gadodiamide administration. Furthermore, INF and signal increase on contrast-enhanced MRI showed high correlation with clinical and histopathological scores. Sensitivities and specificities for detection of moderate and high-grade arthritic joints were slightly lower for NIRF imaging (89%/81%) than for MRI (100%/91%). NIRF imaging using TSC, which is characterized by slower plasma clearance compared to indocyanine green (ICG), has the potential to improve monitoring of inflamed joints.

Gadolinium and 5-Aminolevulinic Acid-induced Protoporphyrin IX Levels in Human Gliomas: An Ex Vivo Quantitative Study to Correlate Protoporphyrin IX Levels and Blood-Brain Barrier Breakdown

PubMed Central

Valdés, Pablo A.; Moses, Ziev B.; Kim, Anthony; Belden, Clifford J.; Wilson, Brian C.; Paulsen, Keith D.; Roberts, David W.; Harris, Brent T.

2012-01-01

In recent years, 5-aminolevulinic acid (ALA)-induced protoporphyrin IX (PpIX) fluorescence guidance has been used as a surgical adjunct to improve the extent of resection of gliomas. Exogenous administration of ALA prior to surgery leads to the accumulation of red fluorescent PpIX in tumor tissue that the surgeon can visualize and thereby discriminate between normal and tumor tissue. Selective accumulation of PpIX has been linked to numerous factors, of which blood-brain barrier (BBB) breakdown has been suggested to be a key factor. To test the hypothesis that PpIX concentration (CPpIX) positively correlates with gadolinium (Gd) concentrations (CGd), we performed ex vivo measurements of PpIX and of Gd using Inductively-Coupled Plasma Mass Spectrometry (ICP-MS) the latter as a quantitative biomarker of BBB breakdown; this was corroborated with immunohistochemistry of microvascular density in surgical biopsies of patients undergoing fluorescence guided surgery for glioma .We found positive correlations between CPpIX and CGd (r = 0.58, p < 0.0001), and between CPpIX and microvascular density (r = 0.55, p < 0.0001), suggesting a significant, yet limited association between BBB breakdown and ALA-induced PpIX fluorescence. To our knowledge, this is the first time that Gd measurements by ICP-MS have been used in human gliomas. PMID:22878664

Fluorescence quenching by TEMPO: a sub-30 A single-molecule ruler.

PubMed

Zhu, Peizhi; Clamme, Jean-Pierre; Deniz, Ashok A

2005-11-01

A series of DNA molecules labeled with 5-carboxytetramethylrhodamine (5-TAMRA) and the small nitroxide radical TEMPO were synthesized and tested to investigate whether the intramolecular quenching efficiency can be used to measure short intramolecular distances in small ensemble and single-molecule experiments. In combination with distance calculations using molecular mechanics modeling, the experimental results from steady-state ensemble fluorescence and fluorescence correlation spectroscopy measurements both show an exponential decrease in the quenching rate constant with the dye-quencher distance in the 10-30 A range. The results demonstrate that TEMPO-5-TAMRA fluorescence quenching is a promising method to measure short distance changes within single biomolecules.

Microvascular Autonomic Composites

DTIC Science & Technology

2012-01-06

thermogravimetric analysis (TGA) was employed. The double wall allowed for increased thermal stability of the microcapsules, which was...fluorescent nanoparticles (Berfield et al. 2006). Digital Image Correlation (DIC) is a data analysis method, which applies a mathematical...Theme IV: Experimental Assessment & Analysis 2.4.1 Optical diagnostics for complex microfluidic systems pg. 50 2.4.2 Fluorescent thermometry

A Practical Teaching Course in Directed Protein Evolution Using the Green Fluorescent Protein as a Model

ERIC Educational Resources Information Center

Ruller, Roberto; Silva-Rocha, Rafael; Silva, Artur; Schneider, Maria Paula Cruz; Ward, Richard John

2011-01-01

Protein engineering is a powerful tool, which correlates protein structure with specific functions, both in applied biotechnology and in basic research. Here, we present a practical teaching course for engineering the green fluorescent protein (GFP) from "Aequorea victoria" by a random mutagenesis strategy using error-prone polymerase…

Iodine imaging in thyroid by fluorescent X-ray CT with 0.05 mm spatial resolution

NASA Astrophysics Data System (ADS)

Takeda, T.; Yu, Q.; Yashiro, T.; Zeniya, T.; Wu, J.; Hasegawa, Y.; Thet-Thet-Lwin; Hyodo, K.; Yuasa, T.; Dilmanian, F. A.; Akatsuka, T.; Itai, Y.

2001-07-01

Fluorescent X-ray computed tomography (FXCT) at a 0.05 mm in-plane spatial resolution and 0.05 mm slice thickness depicted the cross sectional distribution of endogenous iodine within thyroid. The distribution obtained from the FXCT image correlated closely to that obtained from the pathological pictures.

The Application of Fluorescent Quantum Dots to Confocal, Multiphoton, and Electron Microscopic Imaging

PubMed Central

Deerinck, Thomas J.

2009-01-01

Fluorescent quantum dots are emerging as an important tool for imaging cells and tissues, and their unique optical and physical properties have captured the attention of the research community. The most common types of commercially available quantum dots consist of a nanocrystalline semiconductor core composed of cadmium selenide with a zinc sulfide capping layer and an outer polymer layer to facilitate conjugation to targeting biomolecules such as immunoglobulins. They exhibit high fluorescent quantum yields and have large absorption cross-sections, possess excellent photostability, and can be synthesized so that their narrow-band fluorescence emission can occur in a wide spectrum of colors. These properties make them excellent candidates for serving as multiplexing molecular beacons using a variety of imaging modalities including highly correlated microscopies. Whereas much attention has been focused on quantum-dot applications for live-cell imaging, we have sought to characterize and exploit their utility for enabling simultaneous multiprotein immunolabeling in fixed cells and tissues. Considerations for their application to immunolabeling for correlated light and electron microscopic analysis are discussed. PMID:18337229

Probing the conformational dynamics of photosystem I in unconfined and confined spaces.

PubMed

Das, Gaurav; Chattoraj, Shyamtanu; Nandi, Somen; Mondal, Prasenjit; Saha, Abhijit; Bhattacharyya, Kankan; Ghosh, Surajit

2017-12-20

The fluorescence dynamics of Photosystem I (PSI) in bulk water and inside a confined environment like a liposome have been investigated using time resolved confocal microscopy. In bulk water, PSI exhibits a major emission peak at ∼680 nm, while in the liposome it exhibits a markedly blue shifted emission maximum at ∼485 nm. This is indicative of conformational changes due to entrapment and emergence of a stressed conformation of PSI inside the liposome. The observed time constants for the fluorescence lifetime of PSI inside the liposome are significantly high as opposed to PSI in bulk water. More interestingly, the fluorescence intensity of PSI in bulk water exhibits strong fluctuations with many high intensity jumps and these are anti-correlated with the fluorescence lifetime of PSI. In contrast, inside the liposome, no such anti-correlated behaviour is observed. We further demonstrated that PSI exhibits at least two conformational states in bulk water, whereas a single conformation is observed inside the liposome, indicating the conformational rigidity and locking of the PSI complex inside a liposome.

A novel fluorescence microscopy approach to estimate quality loss of stored fruit fillings as a result of browning.

PubMed

Cropotova, Janna; Tylewicz, Urszula; Cocci, Emiliano; Romani, Santina; Dalla Rosa, Marco

2016-03-01

The aim of the present study was to estimate the quality deterioration of apple fillings during storage. Moreover, a potentiality of novel time-saving and non-invasive method based on fluorescence microscopy for prompt ascertainment of non-enzymatic browning initiation in fruit fillings was investigated. Apple filling samples were obtained by mixing different quantities of fruit and stabilizing agents (inulin, pectin and gellan gum), thermally processed and stored for 6-month. The preservation of antioxidant capacity (determined by DPPH method) in apple fillings was indirectly correlated with decrease in total polyphenols content that varied from 34±22 to 56±17% and concomitant accumulation of 5-hydroxymethylfurfural (HMF), ranging from 3.4±0.1 to 8±1mg/kg in comparison to initial apple puree values. The mean intensity of the fluorescence emission spectra of apple filling samples and initial apple puree was highly correlated (R(2)>0.95) with the HMF content, showing a good potentiality of fluorescence microscopy method to estimate non-enzymatic browning. Copyright © 2015 Elsevier Ltd. All rights reserved.

Time-Correlated Single-Photon Counting Fluorescence Imaging of Lipid Domains In Raft-Mimicking Giant Unilamellar Vesicles

NASA Astrophysics Data System (ADS)

Clarke, James; Cheng, Kwan; Shindell, Orrin; Wang, Exing

We have designed and constructed a high-throughput electrofusion chamber and an incubator to fabricate Giant Unilamellar Vesicles (GUVs) consisting of high-melting lipids, low-melting lipids, cholesterol and both ordered and disordered phase sensitive fluorescent probes (DiIC12, dehydroergosterol and BODIPY-Cholesterol). GUVs were formed in a 3 stage pulse sequence electrofusion process with voltages ranging from 50mVpp to 2.2Vpp and frequencies from 5Hz to 10Hz. Steady state and time-correlated single-photon counting (TCSPC) fluorescence lifetime (FLIM) based confocal and/or multi-photon microscopic techniques were used to characterize phase separated lipid domains in GUVs. Confocal imaging measures the probe concentration and the chemical environment of the system. TCSPC techniques determine the chemical environment through the perturbation of fluorescent lifetimes of the probes in the system. The above techniques will be applied to investigate the protein-lipid interactions involving domain formation. Specifically, the mechanisms governing lipid domain formations in the above systems that mimic the lipid rafts in cells will be explored. Murchison Fellowship at Trinity University.

Tracking diurnal changes of photosynthesis and evapotranspiration using fluorescence, gas exchange and hyperspectral remote sensing measurements

NASA Astrophysics Data System (ADS)

Wang, S.; Zhang, L.; Guanter, L.; Huang, C.

2017-12-01

Photosynthesis and evapotranspiration (ET) are the two most important activities of vegetation and make a great contribution to carbon, water and energy exchanges. Remote sensing provides opportunities for monitoring these processes across time and space. This study focuses on tracking diurnal changes of photosynthesis and evapotranspiration over soybean using multiple measurement techniques. Diurnal changes of both remote sensing-based indicators, including active and passive chlorophyll fluorescence and biophysical-related parameters, including photosynthesis rate (photo) and leaf stomatal conductance (cond), were observed. Results showed that both leaf-level steady-state fluorescence (Fs) and canopy-level solar-induced chlorophyll fluorescence were linearly correlated to photosynthetically active radiation (PAR) during the daytime. A double-peak diurnal change curve was observed for leaf-level photo and cond but not for Fs or SIF. Photo and cond showed a strong nonlinear (second-order) correlation, indicating that photosynthesis, which might be remotely sensed by SIF, has the opportunity to track short-term changes of ET. Results presented in this report will be helpful for better understanding the relationship between remote-sensing-based indices and vegetation's biophysical processes.

Chlorophyll Fluorescence as a Possible Tool for Salinity Tolerance Screening in Barley (Hordeum vulgare L.).

PubMed Central

Belkhodja, R.; Morales, F.; Abadia, A.; Gomez-Aparisi, J.; Abadia, J.

1994-01-01

The application of chlorophyll fluorescence measurements to screening barley (Hordeum vulgare L.) genotypes for salinity tolerance has been investigated. Excised barley leaves were cut under water and incubated with the cut end immersed in water or in a 100-mM NaCl solution, either in the dark or in high light. Changes in rapid fluorescence kinetics occurred in excised barley leaves exposed to the saline solution only when the incubation was carried out in the presence of high light. Fluorescence changes consisted of decreases in the variable to maximum fluorescence ratio and in increases in the relative proportion of variable fluorescence leading to point I in the Kautsky fluorescence induction curve. These relative increases in fluorescence at point I appeared to arise from a delayed plastoquinone reoxidation in the dark, since they disappeared after short, far-red illumination, which is known to excite photosystem I preferentially. We show that a significant correlation existed between some fluorescence parameters, measured after a combined salt and high-light treatment, and other independent measurements of salinity tolerance. These results suggest that chlorophyll fluorescence, and especially the relative fluorescence at point I in the Kautsky fluorescence induction curve, could be used for the screening of barley genotypes for salinity tolerance. PMID:12232117

Hessian-based quantitative image analysis of host-pathogen confrontation assays.

PubMed

Cseresnyes, Zoltan; Kraibooj, Kaswara; Figge, Marc Thilo

2018-03-01

Host-fungus interactions have gained a lot of interest in the past few decades, mainly due to an increasing number of fungal infections that are often associated with a high mortality rate in the absence of effective therapies. These interactions can be studied at the genetic level or at the functional level via imaging. Here, we introduce a new image processing method that quantifies the interaction between host cells and fungal invaders, for example, alveolar macrophages and the conidia of Aspergillus fumigatus. The new technique relies on the information content of transmitted light bright field microscopy images, utilizing the Hessian matrix eigenvalues to distinguish between unstained macrophages and the background, as well as between macrophages and fungal conidia. The performance of the new algorithm was measured by comparing the results of our method with that of an alternative approach that was based on fluorescence images from the same dataset. The comparison shows that the new algorithm performs very similarly to the fluorescence-based version. Consequently, the new algorithm is able to segment and characterize unlabeled cells, thus reducing the time and expense that would be spent on the fluorescent labeling in preparation for phagocytosis assays. By extending the proposed method to the label-free segmentation of fungal conidia, we will be able to reduce the need for fluorescence-based imaging even further. Our approach should thus help to minimize the possible side effects of fluorescence labeling on biological functions. © 2017 International Society for Advancement of Cytometry. © 2017 International Society for Advancement of Cytometry.

Interregional synaptic maps among engram cells underlie memory formation.

PubMed

Choi, Jun-Hyeok; Sim, Su-Eon; Kim, Ji-Il; Choi, Dong Il; Oh, Jihae; Ye, Sanghyun; Lee, Jaehyun; Kim, TaeHyun; Ko, Hyoung-Gon; Lim, Chae-Seok; Kaang, Bong-Kiun

2018-04-27

Memory resides in engram cells distributed across the brain. However, the site-specific substrate within these engram cells remains theoretical, even though it is generally accepted that synaptic plasticity encodes memories. We developed the dual-eGRASP (green fluorescent protein reconstitution across synaptic partners) technique to examine synapses between engram cells to identify the specific neuronal site for memory storage. We found an increased number and size of spines on CA1 engram cells receiving input from CA3 engram cells. In contextual fear conditioning, this enhanced connectivity between engram cells encoded memory strength. CA3 engram to CA1 engram projections strongly occluded long-term potentiation. These results indicate that enhanced structural and functional connectivity between engram cells across two directly connected brain regions forms the synaptic correlate for memory formation. Copyright © 2018 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.

Correlated oxygen-sensing PLIM, cell metabolism FLIM and applications

NASA Astrophysics Data System (ADS)

Rück, A. C.; Kalinina, S.; Schäfer, P.; von Einem, B.; von Arnim, C.

2017-02-01

Correlated imaging of phosphorescence and fluorescence lifetime parameters of metabolic markers is a challenge for direct investigating mechanisms related to cell metabolism and oxygen tension. A large variety of clinical phenotypes is associated with mitochondrial defects accomplished with changes in cell metabolism. In many cases the hypoxic microenvironment of cancer cells shifts metabolism from oxidative phosphorylation (OXPHOS) to anaerobic or aerobic glycolysis, a process known as "Warburg" effect. Also during stem cell differentiation a switch in cell metabolism is observed. Mitochondrial dysfunction associated with hypoxia has been invoked in many complex disorders such as type 2 diabetes, Alzheimeŕs disease, cardiac ischemia/reperfusion injury, tissue inflammation and cancer. Cellular responses to oxygen tension have been studied extensively, optical imaging techniques based on time correlated single photon counting (TCSPC) to detect oxygen concentration and distribution are therefore of prominent interest. Moreover, they offer the possibility by inspecting fluorescence decay characteristics of intrinsic coenzymes to directly image metabolic pathways, whereas oxygen tension can be determined by considering the phosphorescence lifetime of a phosphorescent probe. The combination of both fluorescence lifetime imaging (FLIM) of coenzymes like NAD(P)H and FAD and phosphorescence lifetime (PLIM) of phosphorescent dyes could provide valuable information about correlation of metabolic pathways and oxygen tension.

Diketopyrrolopyrrole: brilliant red pigment dye-based fluorescent probes and their applications.

PubMed

Kaur, Matinder; Choi, Dong Hoon

2015-01-07

The development of fluorescent probes for the detection of biologically relevant species is a burgeoning topic in the field of supramolecular chemistry. A number of available dyes such as rhodamine, coumarin, fluorescein, and cyanine have been employed in the design and synthesis of new fluorescent probes. However, diketopyrrolopyrrole (DPP) and its derivatives have a distinguished role in supramolecular chemistry for the design of fluorescent dyes. DPP dyes offer distinctive advantages relative to other organic dyes, including high fluorescence quantum yields and good light and thermal stability. Significant advancements have been made in the development of new fluorescent probes based on DPP in recent years as a result of tireless research efforts by the chemistry scientific community. In this tutorial review, we highlight the recent progress in the development of DPP-based fluorescent probes for the period spanning 2009 to the present time and the applications of these probes to recognition of biologically relevant species including anions, cations, reactive oxygen species, thiols, gases and other miscellaneous applications. This review is targeted toward providing the readers with deeper understanding for the future design of DPP-based fluorogenic probes for chemical and biological applications.

Nanoparticles Incorporated inside Single-Crystals: Enhanced Fluorescent Properties

DOE PAGES

Liu, Yujing; Zang, Huidong; Wang, Ling; ...

2016-09-25

Incorporation of guest materials inside single-crystalline hosts leads to single-crystal composites that have become more and more frequently seen in both biogenic and synthetic crystals. The unique composite structure together with long-range ordering promises special properties that are, however, less often demonstrated. In this study, we examine the fluorescent properties of quantum dots (QDs) and polymer dots (Pdots) encapsulated inside the hosts of calcite single-crystals. Two CdTe QDs and two Pdots are incorporated into growing calcite crystals, as the QDs and Pdots are dispersed in the crystallization media of agarose gels. As a result, enhanced fluorescent properties are obtained frommore » the QDs and Pdots inside calcite single-crystals with greatly improved photostability and significantly prolonged fluorescence lifetime, compared to those in solutions and gels. Particularly, the fluorescence lifetime increases by 0.5-1.6 times after the QDs or Pdots are incorporated. The enhanced fluorescent properties indicate the advantages of encapsulation by single-crystal hosts that provide dense shells to isolate the fluorescent nanoparticles from atmosphere. As such, this work has implications for advancing the research of single-crystal composites toward their functional design.« less

Absorption and fluorescence properties of chromophoric dissolved organic matter: implications for the monitoring of water quality in a large subtropical reservoir.

PubMed

Liu, Xiaohan; Zhang, Yunlin; Shi, Kun; Zhu, Guangwei; Xu, Hai; Zhu, Mengyuan

2014-12-01

The development of techniques for real-time monitoring of water quality is of great importance for effectively managing inland water resources. In this study, we first analyzed the absorption and fluorescence properties in a large subtropical reservoir and then used a chromophoric dissolved organic matter (CDOM) fluorescence monitoring sensor to predict several water quality parameters including the total nitrogen (TN), total phosphorus (TP), chemical oxygen demand (COD), dissolved organic carbon (DOC), and CDOM fluorescence parallel factor analysis (PARAFAC) components in the reservoir. The CDOM absorption coefficient at 254 nm (a(254)), the humic-like component (C1), and the tryptophan-like component (C3) decreased significantly along a gradient from the northwest to the lake center, northeast, southwest, and southeast region in the reservoir. However, no significant spatial difference was found for the tyrosine-like component (C2), which contributed only four marked peaks. A highly significant linear correlation was found between the a(254) and CDOM concentration measured using the CDOM fluorescence sensor (r(2) = 0.865, n = 76, p < 0.001), indicating that CDOM concentrations could act as a proxy for the CDOM absorption coefficient measured in the laboratory. Significant correlations were also found between the CDOM concentration and TN, TP, COD, DOC, and the maximum fluorescence intensity of C1, suggesting that the real-time monitoring of CDOM concentrations could be used to predict these water quality parameters and trace the humic-like fluorescence substance in clear aquatic ecosystems with DOC <2 mg/L and total suspended matter (TSM) concentrations <15 mg/L. These results demonstrate that the CDOM fluorescence sensor is a useful tool for on-line water quality monitoring if the empirical relationship between the CDOM concentration measured using the CDOM fluorescence sensor and the water quality parameters is calibrated and validated.

Estimating the Concentration and Biodegradability of Organic Matter in 22 Wastewater Treatment Plants Using Fluorescence Excitation Emission Matrices and Parallel Factor Analysis

PubMed Central

Yang, Liyang; Shin, Hyun-Sang; Hur, Jin

2014-01-01

This study aimed at monitoring the changes of fluorescent components in wastewater samples from 22 Korean biological wastewater treatment plants and exploring their prediction capabilities for total organic carbon (TOC), dissolved organic carbon (DOC), biochemical oxygen demand (BOD), chemical oxygen demand (COD), and the biodegradability of the wastewater using an optical sensing technique based on fluorescence excitation emission matrices and parallel factor analysis (EEM-PARAFAC). Three fluorescent components were identified from the samples by using EEM-PARAFAC, including protein-like (C1), fulvic-like (C2) and humic-like (C3) components. C1 showed the highest removal efficiencies for all the treatment types investigated here (69% ± 26%–81% ± 8%), followed by C2 (37% ± 27%–65% ± 35%), while humic-like component (i.e., C3) tended to be accumulated during the biological treatment processes. The percentage of C1 in total fluorescence (%C1) decreased from 54% ± 8% in the influents to 28% ± 8% in the effluents, while those of C2 and C3 (%C2 and %C3) increased from 43% ± 6% to 62% ± 9% and from 3% ± 7% to 10% ± 8%, respectively. The concentrations of TOC, DOC, BOD, and COD were the most correlated with the fluorescence intensity (Fmax) of C1 (r = 0.790–0.817), as compared with the other two fluorescent components. The prediction capability of C1 for TOC, BOD, and COD were improved by using multiple regression based on Fmax of C1 and suspended solids (SS) (r = 0.856–0.865), both of which can be easily monitored in situ. The biodegradability of organic matter in BOD/COD were significantly correlated with each PARAFAC component and their combinations (r = −0.598–0.613, p < 0.001), with the highest correlation coefficient shown for %C1. The estimation capability was further enhanced by using multiple regressions based on %C1, %C2 and C3/C2 (r = −0.691). PMID:24448170

An overview of remote sensing of chlorophyll fluorescence

NASA Astrophysics Data System (ADS)

Xing, Xiao-Gang; Zhao, Dong-Zhi; Liu, Yu-Guang; Yang, Jian-Hong; Xiu, Peng; Wang, Lin

2007-03-01

Besides empirical algorithms with the blue-green ratio, the algorithms based on fluorescence are also important and valid methods for retrieving chlorophyll-a concentration in the ocean waters, especially for Case II waters and the sea with algal blooming. This study reviews the history of initial cognitions, investigations and detailed approaches towards chlorophyll fluorescence, and then introduces the biological mechanism of fluorescence remote sensing and main spectral characteristics such as the positive correlation between fluorescence and chlorophyll concentration, the red shift phenomena. Meanwhile, there exist many influence factors that increase complexity of fluorescence remote sensing, such as fluorescence quantum yield, physiological status of various algae, substances with related optical property in the ocean, atmospheric absorption etc. Based on these cognitions, scientists have found two ways to calculate the amount of fluorescence detected by ocean color sensors: fluorescence line height and reflectance ratio. These two ways are currently the foundation for retrieval of chlorophyl l - a concentration in the ocean. As the in-situ measurements and synchronous satellite data are continuously being accumulated, the fluorescence remote sensing of chlorophyll-a concentration in Case II waters should be recognized more thoroughly and new algorithms could be expected.

Fluorescence detection of oral squamous cell carcinoma using Hyperflav

NASA Astrophysics Data System (ADS)

Melnik, Ivan S.; Dets, Sergiy M.; Rawicz, Andrew H.; Zhang, Lewei

2000-05-01

A novel hypericin-based drug HyperflavTM has been evaluated for light-induced fluorescence detection of oral cancer. Squamous cell carcinoma was induced with carcinogenic agent in right pouches of forty hamsters (20/20 males/females). Solution of HyperflavTM was sprinkled into stomach with a single dose 0.2 - 4 mg of pure hypericin per kg b.w. and 4 - 8 hours before fluorescence analysis. In two animal groups with cancer symptoms the autofluorescence and hypericin-induced fluorescence were taken under 442 nm excitation. The buccal mucosa and adjacent areas were measured fiberoptically in-vivo and in-vitro using orange/green ratio (610/540). The in-vivo fluorescence imaging of malignant areas was conducted to assist the biopsy guidance and to compare with white-light images. Histological and morphological analyses were performed from biopsies. Oral squamous cell carcinoma in its early stage demonstrated specific higher 610/540 ratio for 37 tested hamsters. Advanced state involved another higher fluorescence maximum around 640 nm that in our opinion caused by strong porphyrin-induced native fluorescence. Such deformation of fluorescence spectra may lead to inadequate perception of diseased tissue area. To avoid this problem the autofluorescence spectra & images were added. HyperflavTM application is promising for demarcation of early oral cancer when combined with autofluorescence measurements.

Towards Violation of Classical Inequalities using Quantum Dot Resonance Fluorescence

NASA Astrophysics Data System (ADS)

Peiris, Manoj

Self-assembled semiconductor quantum dots have attracted considerable interest recently, ranging from fundamental studies of quantum optics to advanced applications in the field of quantum information science. With their atom-like properties, quantum dot based nanophotonic devices may also substantially contribute to the development of quantum computers. This work presents experimental progress towards the understanding of light-matter interactions that occur beyond well-understood monochromatic resonant light scattering processes in semiconductor quantum dots. First, we report measurements of resonance fluorescence under bichromatic laser excitation. With the inclusion of a second laser, both first-order and second-order correlation functions are substantially altered. Under these conditions, the scattered light exhibits a rich spectrum containing many spectral features that lead to a range of nonlinear multiphoton dynamics. These observations are discussed and compared with a theoretical model. Second, we investigated the light scattered by a quantum dot in the presence of spectral filtering. By scanning the tunable filters placed in front of each detector of a Hanbury-Brown and Twiss setup and recording coincidence measurements, a \\two-photon spectrum" has been experimentally reconstructed for the first time. The two-photon spectrum contains a wealth of information about the cascaded emission involved in the scattering process, such as transitions occurring via virtual intermediate states. Our measurements also reveal that the scattered frequency-filtered light from a quantum dot violates the Cauchy-Schwarz inequality. Finally, Franson-interferometry has been performed using spectrally filtered light from quantum dot resonance fluorescence. Visibilities exceeding the classical limit were demonstrated by using a pair of folded Mach-Zehnder interferometers, paving the way for producing single time-energy entangled photon pairs that could violate Bell's inequalities.

Evaluation of the removal of antiestrogens and antiandrogens via ozone and granular activated carbon using bioassay and fluorescent spectroscopy.

PubMed

Ma, Dehua; Chen, Lujun; Wu, Yuchao; Liu, Rui

2016-06-01

Antiestrogens and antiandrogens are relatively rarely studied endocrine disrupting chemicals which can be found in un/treated wastewaters. Antiestrogens and antiandrogens in the wastewater treatment effluents could contribute to sexual disruption of organisms. In this study, to assess the removal of non-specific antiestrogens and antiandrogens by advanced treatment processes, ozonation and adsorption to granular activated carbon (GAC), the biological activities and excitation emission matrix fluorescence spectroscopy of wastewater were evaluated. As the applied ozone dose increased to 12 mg/L, the antiestrogenic activity dramatically decreased to 3.2 μg 4-hydroxytamoxifen equivalent (4HEQ)/L, with a removal efficiency of 84.8%, while the antiandrogenic activity was 23.1 μg flutamide equivalent (FEQ)/L, with a removal efficiency of 75.5%. The removal of antiestrogenic/antiandrogenic activity has high correlation with the removal of fulvic acid-like materials and humic acid-like organics, suggesting that they can be used as surrogates for antiestrogenic/antiandrogenic activity during ozonation. The adsorption kinetics of antiestrogenic activity and antiandrogenic activity were well described by pseudo-second-order kinetics models. The estimated equilibrium concentration of antiestrogenic activity is 7.9 μg 4HEQ/L with an effective removal efficiency of 70.5%, while the equilibrium concentration of antiandrogenic activity is 33.7 μg FEQ/L with a removal efficiency of 67.0%. Biological activity evaluation of wastewater effluents is an attractive way to assess the removal of endocrine disrupting chemicals by different treatment processes. Fluorescence spectroscopy can be used as a surrogate measure of bioassays during ozonation. Copyright © 2016 Elsevier Ltd. All rights reserved.

Two-photon excited fluorescence spectroscopy and imaging of melanin in vitro and in vivo

NASA Astrophysics Data System (ADS)

Krasieva, Tatiana B.; Liu, Feng; Sun, Chung-Ho; Kong, Yu; Balu, Mihaela; Meyskens, Frank L.; Tromberg, Bruce J.

2012-03-01

The ability to detect early melanoma non-invasively would improve clinical outcome and reduce mortality. Recent advances in two-photon excited fluorescence (TPEF) in vivo microscopy offer a powerful tool in early malignant melanoma diagnostics. The goal of this work was to develop a TPEF optical index for measuring relative concentrations of eumelanin and pheomelanin since ex vivo studies show that changes in this ratio have been associated with malignant transformation. We acquired TPEF emission spectra (λex=1000 nm) of melanin from several specimens, including human hair, malignant melanoma cell lines, and normal melanocytes and keratinocytes in different skin layers (epidermis, papillary dermis) in five healthy volunteers in vivo. We found that the pheomelanin emission peaks at around 620 nm and is blue-shifted from the eumelanin with broad maximum at 640-680nm. We defined "optical melanin index" (OMI) as a ratio of fluorescence signal intensities measured at 645 nm and 615nm. The measured OMI for a melanoma cell line MNT-1 was 1.6+/-0.2. The MNT-46 and MNT-62 lines (Mc1R gene knockdown) showed an anticipated change in melanins production ratio and had OMI of 0.55+/-0.05 and 0.17+/-0.02, respectively, which strongly correlated with HPLC data obtained for these lines. Average OMI measured for basal cells layers (melanocytes and keratinocytes) in normal human skin type I, II-III (not tanned and tanned) in vivo was 0.5, 1.05 and 1.16 respectively. We could not dependably detect the presence of pheomelanin in highly pigmented skin type V-VI. These data suggest that a non-invasive TPEF index could potentially be used for rapid melanin ratio characterization both in vitro and in vivo, including pigmented lesions.

Passive and active targeting of quantum dots for whole-body fluorescence imaging of breast cancer xenografts.

PubMed

Balalaeva, Irina V; Zdobnova, Tatiana A; Krutova, Irina V; Brilkina, Anna A; Lebedenko, Ekaterina N; Deyev, Sergey M

2012-11-01

Far-red and near-infrared fluorescent quantum dots (QDs) have become advancing contrast agents for efficient whole-body tumor imaging. In this study, we investigated the possibility of the vital fluorescence imaging of tumor using two contrast agents on the basis of QDs: bioinert QDs coated with polyethyleneglycol and QDs bound with anti-HER2/neu scFv antibodies. HER2/neu-positive breast cancer tumor xenografts in nude mice were used as a model. It was shown that both bioinert and tumor-targeted QD probes can be successfully applied for visualization of the tumor using in vivo imaging method, but fluorescent signal of QD-4D5scFv in tumors was considerably stronger than that of QD-PEG. Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Quantitative segmentation of fluorescence microscopy images of heterogeneous tissue: Approach for tuning algorithm parameters

NASA Astrophysics Data System (ADS)

Mueller, Jenna L.; Harmany, Zachary T.; Mito, Jeffrey K.; Kennedy, Stephanie A.; Kim, Yongbaek; Dodd, Leslie; Geradts, Joseph; Kirsch, David G.; Willett, Rebecca M.; Brown, J. Quincy; Ramanujam, Nimmi

2013-02-01

The combination of fluorescent contrast agents with microscopy is a powerful technique to obtain real time images of tissue histology without the need for fixing, sectioning, and staining. The potential of this technology lies in the identification of robust methods for image segmentation and quantitation, particularly in heterogeneous tissues. Our solution is to apply sparse decomposition (SD) to monochrome images of fluorescently-stained microanatomy to segment and quantify distinct tissue types. The clinical utility of our approach is demonstrated by imaging excised margins in a cohort of mice after surgical resection of a sarcoma. Representative images of excised margins were used to optimize the formulation of SD and tune parameters associated with the algorithm. Our results demonstrate that SD is a robust solution that can advance vital fluorescence microscopy as a clinically significant technology.

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.