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A NOVEL SETUP FOR HIGH-PRESSURE RAMAN SPECTROSCOPY UNDER A MICROSCOPE
Title
A NOVEL SETUP FOR HIGH-PRESSURE RAMAN SPECTROSCOPY UNDER A MICROSCOPE.
Creator
Oakeson, Thomas, Schulte, Alfons, University of Central Florida
Abstract / Description
Functional properties of biological molecules and cells are affected by environmental parameters such as temperature and pressure. While Raman spectroscopy provides an intrinsic probe of molecular structural changes, the incorporation of a microscope enables studies of minuscule amounts of biological compounds with spatial resolution on a micron scale. We have developed a novel setup which combines a Raman microscope and a high pressure cell. A micro-capillary made out of fused silica...
Show moreFunctional properties of biological molecules and cells are affected by environmental parameters such as temperature and pressure. While Raman spectroscopy provides an intrinsic probe of molecular structural changes, the incorporation of a microscope enables studies of minuscule amounts of biological compounds with spatial resolution on a micron scale. We have developed a novel setup which combines a Raman microscope and a high pressure cell. A micro-capillary made out of fused silica simultaneously serves as the supporting body and the optical window of the pressure cell. The cell has been tested over the pressure range from 0.1 to 4 kbar. Raman spectra of less than 100 nanoliter amount of amino acid and protein solutions have been measured in the micro-capillary high pressure cell. It is also demonstrated that the setup is well suited for spectrally resolved fluorescence measurements at variable pressure.
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Date Issued
2007
Identifier
CFE0001683, ucf:47208
Format
Document (PDF)
PURL
http://purl.flvc.org/ucf/fd/CFE0001683
In Actu Et In Silicio: Linear and Nonlinear Photophysical Characterization of a Novel Europium Complex, and Incorporating Computational Calculations in the Analysis of Novel Organic Compounds
Title
In Actu Et In Silicio: Linear and Nonlinear Photophysical Characterization of a Novel Europium Complex, and Incorporating Computational Calculations in the Analysis of Novel Organic Compounds.
Creator
Woodward, Adam, Belfield, Kevin, Campiglia, Andres, Harper, James, Frazer, Andrew, Cheng, Zixi, University of Central Florida
Abstract / Description
Despite not being a tangible substance, light is becoming an increasingly valuable tool in numerous areas of science and technology: the use of laser excitation of a fluorescent probe can generate incredibly detailed images of cellular structures without the need for large amounts of dissection; new types of solar cells are being produced using organic dyes to harvest light; computer data can be stored by inducing a chemical change in a compound through irradiation with light. However, before...
Show moreDespite not being a tangible substance, light is becoming an increasingly valuable tool in numerous areas of science and technology: the use of laser excitation of a fluorescent probe can generate incredibly detailed images of cellular structures without the need for large amounts of dissection; new types of solar cells are being produced using organic dyes to harvest light; computer data can be stored by inducing a chemical change in a compound through irradiation with light. However, before any of these materials can be applied in such a way, their properties must first be analyzed for them to be deemed viable.The focus of this dissertation is the photophysical characterization, linear and nonlinear, of a several novel organic compounds, and a europium complex, as well as using quantum chemical calculation techniques to understand some of the phenomena that are witnessed and begin to develop predictive capability. The nonlinear characterization of compounds utilizes wavelengths outside of their linear absorption range, where a focused beam can achieve the same excitation as one at half the wavelength, though this effect has a quadratic dependence on power.The potential for nonlinear excitation, or two-photon absorption (2PA), is becoming of increasing interest and importance for organic chromophores. Exciting only a small volume of material at a focal point makes it possible to nondestructively image samples in 3-dimensions, record data in multiple layers, and fabricate intricate structures through photopolymerization reactions.Lanthanides such as europium are known to exhibit sharp emission bands when excited, typically through an antenna effect due to the low probability of achieving direct excitation. This emission is long-lived, and through gating systems can readily be separated from background noise and autofluorescence (often observed in biological samples) that have much shorter lifetimes. Thus, one of the foci of this dissertation is the photophysical investigation of a series of novel lanthanide complexes, with particular attention to a europium complex.
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Date Issued
2014
Identifier
CFE0005908, ucf:50891
Format
Document (PDF)
PURL
http://purl.flvc.org/ucf/fd/CFE0005908
Solid Phase Extraction Room Temperature Fluorescence Spectroscopy for the Direct Quantification of Monohydroxy Metabolites of Polycyclic Aromatic Hydrocarbons in Urine Samples
Title
Solid Phase Extraction Room Temperature Fluorescence Spectroscopy for the Direct Quantification of Monohydroxy Metabolites of Polycyclic Aromatic Hydrocarbons in Urine Samples.
Creator
Calimag, Korina Jesusa, Campiglia, Andres, Belfield, Kevin, Yestrebsky, Cherie, Chumbimuni Torres, Karin, Schulte, Alfons, University of Central Florida
Abstract / Description
Polycyclic aromatic hydrocarbons (PAH) are important environmental pollutants originating from a wide variety of natural and anthropogenic sources. Because many of them are highly suspect as etiological agents in human cancer, chemical analysis of PAH is of great environmental and toxicological importance. Current methodology for PAH follows the classical pattern of sample preparation and chromatographic analysis. Sample preparation pre-concentrates PAH, simplifies matrix composition, and...
Show morePolycyclic aromatic hydrocarbons (PAH) are important environmental pollutants originating from a wide variety of natural and anthropogenic sources. Because many of them are highly suspect as etiological agents in human cancer, chemical analysis of PAH is of great environmental and toxicological importance. Current methodology for PAH follows the classical pattern of sample preparation and chromatographic analysis. Sample preparation pre-concentrates PAH, simplifies matrix composition, and facilitates analytical resolution in the chromatographic column. Among the several approaches that exist to pre-concentrate PAH from water samples, the Environmental Protection Agency (EPA) recommends the use of solid-phase extraction (SPE). High-performance liquid chromatography (HPLC) and gas chromatography-mass spectrometry (GC-MS) are the basis for standard PAH identification and determination. Ultraviolet (UV) absorption and room temperature fluorescence detection are both widely used in HPLC, but the specificity of these detectors is modest. Since PAH identifi(&)#172;cation is solely based on retention times, unambiguous PAH identification requires complete chromatographic resolution of sample components. When HPLC is applied to (")unfamiliar(") samples, the EPA recommends that a supporting analytical technique such as GC-MS be applied to verify compound identification and to check peak-purity HPLC fractions. Independent of the volume of extracted water, the approximate time required to separate and determine the sixteen (")priority pollutants(") (EPA-PAH) via HPLC is approximately 60min. If additional GC-MS analysis is required for unambiguous PAH determination, the total analysis time will reach 2-3 hours per sample. If the concentrations of target species are found to lie outside the detector's response range, the sample must be diluted and the process repeated. These are important considerations when routine analysis of numerous samples is contemplated. Parent PAH are relatively inert and need metabolic activation to express their carcinogenicity. By virtue of the rich heterogeneous distribution of metabolic products they produce, PAH provide a full spectrum of the complexity associated with understanding the initial phase of carcinogenesis. PAH metabolites include a variety of products such as expoxides, hydroxyl aromatics, quinines, dihydrodiols, dioepoxides, tetrols and water soluble conjugates. During the past decades tremendous efforts have been made to develop bio-analytical techniques that possess the selectivity and sensitivity for the problem at hand. Depending on the complexity of the sample and the relative concentrations of the targeted metabolites, a combination of sample preparation techniques is often necessary to reach the limits of detection of the instrumental method of analysis. The numerous preparation steps open ample opportunity to metabolite loss and collection of inaccurate data. Separation of metabolites has been accomplished via HPLC, capillary electrophoresis (CE) and GC-MS. Unfortunately, the existence of chemically related metabolic products with virtually identical fragmentation patterns often challenges the specificity of these techniques. This dissertation presents significant improvements in various fronts. Its first original component (-) which we have named solid-phase nano-extraction (SPNE) - deals with the use of gold nanoparticles (Au NPs) as extracting material for PAH. The advantages of SPNE are demonstrated for the analysis of PAH in water samples via both HPLC1 and Laser-Excited Time-Resolved Shpol'skii Spectroscopy (LETRSS).2 The same concept is then extended to the analysis of monohydroxy-PAH in urine samples via SPE- HPLC3 and In-Capillary SPNE-CE.4 The second original component of this dissertation describes the application of Shpol'skii Spectroscopy to the analysis of polar PAH metabolites. The outstanding selectivity and sensitivity for the direct analysis of PAH at trace concentration levels has made Shpol'skii spectroscopy a leading technique in environmental analysis.5 Unfortunately, the requirement of a specific guest-host combination - typically a non-polar PAH dissolved in an n-alkane - has hindered its widespread application to the field of analytical chemistry. This dissertation takes the first steps in removing this limitation demonstrating its feasibility for the analysis of polar benzo[a]pyrene metabolites in alcohol matrixes.
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Date Issued
2013
Identifier
CFE0005141, ucf:50693
Format
Document (PDF)
PURL
http://purl.flvc.org/ucf/fd/CFE0005141
UP-CONVERSION IN RARE-EARTH DOPED MICRO-PARTICLES APPLIED TO NEW EMISSIVE 2D DISLAYS
Title
UP-CONVERSION IN RARE-EARTH DOPED MICRO-PARTICLES APPLIED TO NEW EMISSIVE 2D DISLAYS.
Creator
Milliez, Anne, Bass, Michael, University of Central Florida
Abstract / Description
Up-conversion (UC) in rare-earth co-doped fluorides to convert diode laser light in the near infrared to red, green and blue visible light is applied to make possible high performance emissive displays. The infrared-to-visible UC in the materials we study is a sequential form of non-linear two photon absorption in which a strong absorbing constituent absorbs two low energy photons and transfers this energy to another constituent which emits visible light. Some of the UC emitters' most...
Show moreUp-conversion (UC) in rare-earth co-doped fluorides to convert diode laser light in the near infrared to red, green and blue visible light is applied to make possible high performance emissive displays. The infrared-to-visible UC in the materials we study is a sequential form of non-linear two photon absorption in which a strong absorbing constituent absorbs two low energy photons and transfers this energy to another constituent which emits visible light. Some of the UC emitters' most appealing characteristics for displays are: a wide color gamut with very saturated colors, very high brightness operation without damage to the emitters, long lifetimes and efficiencies comparable to those of existing technologies. Other advantages include simplicity of fabrication, versatility of operating modes, and the potential for greatly reduced display weight and depth. Thanks to recent advances in material science and diode laser technology at the excitation wavelength, UC selected materials can be very efficient visible emitters. However, optimal UC efficiencies strongly depend on chosing proper operating conditions. In this thesis, we studied the conditions required for optimization. We demonstrated that high efficiency UC depends on high pump irradiance, low temperature and low scattering. With this understanding we can predict how to optimally use UC emitters in a wide range of applications. In particular, we showed how our very efficient UC emitters can be applied to make full color displays and very efficient white light sources.
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Date Issued
2006
Identifier
CFE0001058, ucf:46828
Format
Document (PDF)
PURL
http://purl.flvc.org/ucf/fd/CFE0001058
ROLE OF MEMBRANE LIPIDS IN MODULATING PROTEIN STRUCTURE & FUNCTION
Title
ROLE OF MEMBRANE LIPIDS IN MODULATING PROTEIN STRUCTURE & FUNCTION.
Creator
Ray, Supriyo, Tatulian, Suren, University of Central Florida
Abstract / Description
A-B family of toxins consists of plant toxins such as ricin and bacterial toxins such as cholera. The A subunit is the enzymatic domain and the B subunit is the receptor binding domain. Commonly, these toxins bind to the target cell plasma membrane receptors through their B subunit followed by endocytosis and a transport to the endoplasmic reticulum (ER). Inside the ER, the A subunit dissociates from the rest of the toxin, unfolds and triggers the ER quality control mechanism of ER-associated...
Show moreA-B family of toxins consists of plant toxins such as ricin and bacterial toxins such as cholera. The A subunit is the enzymatic domain and the B subunit is the receptor binding domain. Commonly, these toxins bind to the target cell plasma membrane receptors through their B subunit followed by endocytosis and a transport to the endoplasmic reticulum (ER). Inside the ER, the A subunit dissociates from the rest of the toxin, unfolds and triggers the ER quality control mechanism of ER-associated degradation (ERAD). Most ERAD substrates are purged out of the ER into the cytosol for proteasomal degradation. However, the low content of lysine amino acid residues allows the toxin to evade polyubiquitination and subsequent proteasomal degradation. The toxin A subunit refolds into an active conformation in the cytosol, setting off downstream toxic events. In the first part of my thesis, the hypothesis was tested that inhibiting the unfolding of the toxin A subunit inside the ER will prevent ERAD activation, toxin export to the cytosol and intoxication. The chemical chaperones glycerol and sodium 4-phenyl butyrate (PBA) were used to inhibit the toxin A chain unfolding. In vitro biophysical experiments indicated that both chemical chaperones indeed stabilize the cholera toxin A subunit and prevent cytotoxicity. In case of ricin, both chaperones stabilized the toxin A chain but only glycerol prevented cytotoxicity. Additional experiments showed that PBA-treated ricin A chain is destabilized when exposed to anionic lipid membranes mimicking the properties of the ER membrane. In contrast, anionic lipid did not prevent ricin A chain stabilization by glycerol. This explains why glycerol but not PBA blocked ricin intoxication, as only glycerol stabilizes ricin A chain in the presence of ER membranes. Cholera toxin in contrast, remained either unaffected or slightly stabilized in presence of anionic lipids both in presence and absence of PBA. This shows that destabilization by anionic lipids is a toxin-specific rather than a general effect. In the second part of my thesis, the effect of inner leaflet of plasma membrane on the structure of cholera toxin A chain (CTA1) was studied. Since CTA1 refolds into an active conformation in the cytosol in association with unidentified host factors, I hypothesized that inner leaflet of the plasma membrane might play a role to stabilization and/or refolding of CTA1. CTA1 was shown to be a membrane interacting protein, and membranes mimicking lipid rafts had a significant stabilizing effect on its structure. Lipid rafts helped in the regaining of the tertiary and secondary structure of CTA1, while non-raft lipids had a smaller stabilizing effect on CTA1 structure. In the next part of my thesis, I studied the effect of membrane binding on the structure and function of human pancreatic phospholipase A2 (PLA2). Lipid thermal phase transition was found to have a dramatic effect on PLA2 activity. It was also established that although membrane binding and insertion was essential for of PLA2 activity, lipid structural heterogeneity was more important than the depth of membrane insertion for enzyme activation. Most importantly, significant changes in PLA2 secondary and tertiary structures were identified that evidently contribute to the interfacial activation of PLA2. Overall, we conclude that the function of membrane binding enzymes can be significantly modulated via conformational changes induced by interactions with membranes. Thus, we have elucidated various roles of membrane lipids from unfolding and refolding to activation and modulation of membrane binding enzymes. Physical properties of lipids help in regulating various aspects of protein structure and function and their analysis helped us in appreciating the influence wielded by the membrane lipids in the enzyme's surrounding environment.
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Date Issued
2011
Identifier
CFE0004035, ucf:49184
Format
Document (PDF)
PURL
http://purl.flvc.org/ucf/fd/CFE0004035
Improvements on Instrumentation to Explore the Multidimensionality of Luminescence Spectroscopy
Title
Improvements on Instrumentation to Explore the Multidimensionality of Luminescence Spectroscopy.
Creator
Moore, Anthony, Campiglia, Andres, Chumbimuni Torres, Karin, Harper, James, Rex, Matthew, Lee, Woo Hyoung, University of Central Florida
Abstract / Description
This dissertation presents experimental and instrumentation developments that take full advantage of the multidimensional nature of line narrowing spectroscopy at liquid nitrogen (77 K) and liquid helium (4.2 K) temperatures. The inconvenience of sample freezing procedures is eliminated with the aid of cryogenic fiber optic probes. Rapid collection of multidimensional data formats such as wavelength time matrices, excitation emission matrices, time-resolved excitation emission matrices and...
Show moreThis dissertation presents experimental and instrumentation developments that take full advantage of the multidimensional nature of line narrowing spectroscopy at liquid nitrogen (77 K) and liquid helium (4.2 K) temperatures. The inconvenience of sample freezing procedures is eliminated with the aid of cryogenic fiber optic probes. Rapid collection of multidimensional data formats such as wavelength time matrices, excitation emission matrices, time-resolved excitation emission matrices and time resolved excitation emission cubes is made possible with the combination of a pulsed tunable dye laser, a spectrograph and an intensifier-charged coupled device. These data formats provide unique opportunities for processing vibrational luminescence data with second order multivariate calibration algorithms. The use of cryogenic fiber optic probes is extended to commercial instrumentation. An attractive feature of spectrofluorimeters with excitation and emission monochromators is the possibility to record synchronous spectra. The advantages of this approach, which include narrowing of spectral bandwidth and simplification of emission spectra, were demonstrated with the direct analysis of highly toxic dibenzopyrene isomers. The same is true for the collection of steady-state fluorescence excitation-emission matrices. These approaches provide a general solution to unpredictable spectral interference, a ubiquitous problem for the analysis of organic pollutants in environmental samples of unknown composition. Since commercial spectrofluorimeters are readily available in most academic institutions, industrial settings and research institutes, the developments presented here should facilitate the widespread application of line-narrowing spectroscopic techniques to the direct determination, no chromatographic separation, of highly toxic compounds in complex environmental matrixes of unknown composition.
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Date Issued
2015
Identifier
CFE0005847, ucf:50934
Format
Document (PDF)
PURL
http://purl.flvc.org/ucf/fd/CFE0005847
NOVEL IMPROVEMENTS ON THE ANALYTICAL CHEMISTRY OF POLYCYCLIC AROMATIC HYDROCARBONS AND THEIR METABOLITES
Title
NOVEL IMPROVEMENTS ON THE ANALYTICAL CHEMISTRY OF POLYCYCLIC AROMATIC HYDROCARBONS AND THEIR METABOLITES.
Creator
Wang, Huiyong, Campiglia, Andres, University of Central Florida
Abstract / Description
Polycyclic aromatic hydrocarbons (PAH) are important environmental pollutants originating from a wide variety of natural and anthropogenic sources. Because many of them are highly suspect as etiological agents in human cancer, chemical analysis of PAH is of great environmental and toxicological importance. Current methodology for PAH follows the classical pattern of sample preparation and chromatographic analysis. Sample preparation pre-concentrates PAH, simplifies matrix composition, and...
Show morePolycyclic aromatic hydrocarbons (PAH) are important environmental pollutants originating from a wide variety of natural and anthropogenic sources. Because many of them are highly suspect as etiological agents in human cancer, chemical analysis of PAH is of great environmental and toxicological importance. Current methodology for PAH follows the classical pattern of sample preparation and chromatographic analysis. Sample preparation pre-concentrates PAH, simplifies matrix composition, and facilitates analytical resolution in the chromatographic column. Among the several approaches that exist to pre-concentrate PAH from water samples, the Environmental Protection Agency (EPA) recommends the use of solid-phase extraction (SPE). High-performance liquid chromatography (HPLC) and gas chromatography-mass spectrometry (GC-MS) are the basis for standard PAH identification and determination. Ultraviolet (UV) absorption and room temperature fluorescence detection are both widely used in HPLC, but the specificity of these detectors is modest. Since PAH identification is solely based on retention times, unambiguous PAH identification requires complete chromatographic resolution of sample components. When HPLC is applied to "unfamiliar" samples, the EPA recommends that a supporting analytical technique such as GC-MS be applied to verify compound identification and to check peak-purity HPLC fractions. Independent of the volume of extracted water, the approximate time required to separate and determine the sixteen "priority pollutants" (EPA-PAH) via HPLC is approximately 60min. If additional GC-MS analysis is required for unambiguous PAH determination, the total analysis time will reach 2-3 hours per sample. If the concentrations of target species are found to lie outside the detector's response range, the sample must be diluted and the process repeated. These are important considerations when routine analysis of numerous samples is contemplated. Parent PAH are relatively inert and need metabolic activation to express their carcinogenicity. By virtue of the rich heterogeneous distribution of metabolic products they produce, PAH provide a full spectrum of the complexity associated with understanding the initial phase of carcinogenesis. PAH metabolites include a variety of products such as expoxides, hydroxyl aromatics, quinines, dihydrodiols, dioepoxides, tetrols and water soluble conjugates. During the past decades tremendous efforts have been made to develop bio-analytical techniques that possess the selectivity and sensitivity for the problem at hand. Depending on the complexity of the sample and the relative concentrations of the targeted metabolites, a combination of sample preparation techniques is often necessary to reach the limits of detection of the instrumental method of analysis. The numerous preparation steps open ample opportunity to metabolite loss and collection of inaccurate data. Separation of metabolites has been accomplished via HPLC, capillary electrophoresis (CE) and GC-MS. Unfortunately, the existence of chemically related metabolic products with virtually identical fragmentation patterns often challenges the specificity of these techniques. This dissertation presents significant improvements in various fronts. Its first original component - which we have named solid-phase nano-extraction (SPNE) - deals with the use of gold nanoparticles (Au NPs) as extracting material for PAH. The advantages of SPNE are demonstrated for the analysis of PAH in water samples via both HPLC and Laser-Excited Time-Resolved Shpol'skii Spectroscopy (LETRSS). The same concept is then extended to the analysis of monohydroxy-PAH in urine samples via SPE- HPLC and In-Capillary SPNE-CE. The second original component of this dissertation describes the application of Shpol'skii Spectroscopy to the analysis of polar PAH metabolites. The outstanding selectivity and sensitivity for the direct analysis of PAH at trace concentration levels has made Shpol'skii spectroscopy a leading technique in environmental analysis. Unfortunately, the requirement of a specific guest-host combination - typically a non-polar PAH dissolved in an n-alkane - has hindered its widespread application to the field of analytical chemistry. This dissertation takes the first steps in removing this limitation demonstrating its feasibility for the analysis of polar benzopyrene metabolites in alcohol matrixes.
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Date Issued
2010
Identifier
CFE0003202, ucf:48579
Format
Document (PDF)
PURL
http://purl.flvc.org/ucf/fd/CFE0003202
Fluorescence Lifetime Imaging and Spectroscopy Aided Tracking of ZnO and CdS:Mn/ZnS/ N-acetyl cysteine (NAC) Quantum Dots in Citrus Plants
Title
Fluorescence Lifetime Imaging and Spectroscopy Aided Tracking of ZnO and CdS:Mn/ZnS/ N-acetyl cysteine (NAC) Quantum Dots in Citrus Plants.
Creator
Washington, Torus, Gesquiere, Andre, Rajaraman, Swaminathan, Zhai, Lei, University of Central Florida
Abstract / Description
In this thesis, we present an efficacious way of tracking nanoparticle movement in plant tissue through the use of fluorescence lifetime imaging (FLIM) and spectroscopy as well as a review of nanoparticle uptake in plants and the proposed mechanisms governing them. Given the increasing number of nanomaterials in agriculture and society as a whole, proper imaging tools and proactive measures must be taken to track nanoparticle movement in plant tissues and create infrastructure and products to...
Show moreIn this thesis, we present an efficacious way of tracking nanoparticle movement in plant tissue through the use of fluorescence lifetime imaging (FLIM) and spectroscopy as well as a review of nanoparticle uptake in plants and the proposed mechanisms governing them. Given the increasing number of nanomaterials in agriculture and society as a whole, proper imaging tools and proactive measures must be taken to track nanoparticle movement in plant tissues and create infrastructure and products to keep things sustainable and safe. Herein we report a ZnO comparable nanoparticle(-) a CdS:Mn/ZnS/ N-acetyl cysteine (NAC) quantum dot(-) which boasts longer lifetimes and suitable fluorescent properties above ZnO to properly delineate from plant tissue fluorescence of chlorophyll and cinnamic acids. In addition to FLIM mapping, quantum dot localization in plant vascular tissue was clearly seen and confirmed via characteristic emission spectra and time correlated single photon counting decay curves (TCSPC). Most quantum dots were seen to reside in the xylem. Plant age and structure was seen to affect uptake. QD size likely restricted extensive translocation. Inhibitive effects of QDs were likely water and mechanical stress. We surmise that travel of the cadmium quantum dots up the leaf and branch plant tissues is likely most governed by diffusion as the quantum dots bound to the cell structures create a diffusion gradient which aids travel up the leaf.
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Date Issued
2017
Identifier
CFE0006820, ucf:51772
Format
Document (PDF)
PURL
http://purl.flvc.org/ucf/fd/CFE0006820