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CONFOCAL LASER SCANNING MICROSCOPY AS A TOOL FOR THE INVESTIGATION OF TETRACYCLINE FLUORESCENCE IN ARCHAEOLOGICALHUMAN BONE
Title
CONFOCAL LASER SCANNING MICROSCOPY AS A TOOL FOR THE INVESTIGATION OF TETRACYCLINE FLUORESCENCE IN ARCHAEOLOGICALHUMAN BONE.
Creator
Maggiano, Corey, Waterman, Jane, University of Central Florida
Abstract / Description
Fluorochromes such as tetracycline have been used to label bone for histomorphometric analysis, measuring bone formation, growth, maintenance, and pathology. More recently, similar fluorescence has been observed in ancient human bone. Attributed to tetracycline (TC) exposure, this phenomenon could affect various aspects of health during life and/or preservation of remains postmortem. Standard epifluorescence microscopy is the most common tool employed in the analysis of these labels. Though...
Show moreFluorochromes such as tetracycline have been used to label bone for histomorphometric analysis, measuring bone formation, growth, maintenance, and pathology. More recently, similar fluorescence has been observed in ancient human bone. Attributed to tetracycline (TC) exposure, this phenomenon could affect various aspects of health during life and/or preservation of remains postmortem. Standard epifluorescence microscopy is the most common tool employed in the analysis of these labels. Though valuable, this technique is limited by its inability to penetrate bone three-dimensionally and its inclusion of out-of-focus light, possibly disrupting accurate analysis. Confocal Laser Scanning Microscopy (CLSM) has been demonstrated as a valuable tool for three-dimensional histology. Its application to the study of compact bone fluorescence has been lacking, especially in archaeological and forensic sciences. In the following two papers, modern TC-controlled bone is compared to well preserved archaeological bone recovered from the Dakhleh Oasis, Egypt, using both standard wide-field and more modern confocal techniques for imaging and analysis. Spectral analysis via CLSM shows that both modern and ancient fluorescent labels in bone share the exact same fluorescence emission peak at 525 nm. Differences in the shape of the spectral curve and photobleaching characteristics are discussed. In addition, CLSM's high-resolution two- and three-dimensional imaging capabilities (in polarized light, scattered light, and fluorescence light) are found to increase the flexibility and creativity of investigations into the occurrence of tetracycline labels in archaeological bone and could have added benefits for modern medical and anatomical experimentation.
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Date Issued
2005
Identifier
CFE0000836, ucf:46680
Format
Document (PDF)
PURL
http://purl.flvc.org/ucf/fd/CFE0000836
Multidimensional Room-Temperature Fluorescence Microscopy for the Nondestructive Analysis of Forensic Trace Textile Fibers
Title
Multidimensional Room-Temperature Fluorescence Microscopy for the Nondestructive Analysis of Forensic Trace Textile Fibers.
Creator
Mujumdar, Nirvani, Campiglia, Andres, Sigman, Michael, Harper, James, Rex, Matthew, Peale, Robert, University of Central Florida
Abstract / Description
The purpose of this dissertation is to advance nondestructive methodology for forensic fiber examination. Non-destructive techniques that can either discriminate between similar fibers or match a known to a questioned fiber (-) and still preserve the physical integrity of the fibers for further court examination - are highly valuable in forensic science. A challenging aspect of forensic fiber examinations involves the comparison of fibers colored with visually indistinguishable dyestuffs....
Show moreThe purpose of this dissertation is to advance nondestructive methodology for forensic fiber examination. Non-destructive techniques that can either discriminate between similar fibers or match a known to a questioned fiber (-) and still preserve the physical integrity of the fibers for further court examination - are highly valuable in forensic science. A challenging aspect of forensic fiber examinations involves the comparison of fibers colored with visually indistinguishable dyestuffs. This is not an uncommon situation, as there are numerous indistinguishable fibers pre-dyed with commercial dyes of virtually identical colors. Minimal chemical structural variations are actually encouraged by the dye patent process and commercial competition.The common denominator to forensic methodology is the fact that fiber analysis primarily focuses on the dyes used to color the fibers and do not investigate other potential discriminating components present in the fiber. This dissertation explores a different aspect of fiber analysis as it focuses on the total fluorescence emission of fibers. In addition to the contribution of the textile dye (or dyes) to the fluorescence spectrum of the fiber, we consider the contribution of intrinsic fluorescence impurities (-) i.e. impurities imbedded into the fibers during fabrication of garments - as a reproducible source of fiber comparison. Although fluorescence microscopy is used in forensic labs for single fiber examination, measurements are made with the aid of band-pass filters that provide very limited information on the spectral profiles of fibers. We take the non-destructive nature of fluorescence microscopy to a higher level of selectivity with the collection of room-temperature fluorescence excitation emission matrices (RTF-EEMs). The information contained in the EEMs was first used to select the best excitation wavelength for recording first order data, i.e. two-dimensional fluorescence spectra. Pairwise comparisons involved the following visually indistinguishable fibers: nylon 361 pre-dyed with acid yellow (AY) 17 and AY 23, acrylic 864 pre-dyed with basic green (BG) 1 and BG 4, acetate satin 105B pre-dyed with disperse blue (DB) 3 and DB 14, and polyester 777 pre-dyed with disperse red (DR) 1 and DR 19. With the exception of acrylic 864 fibers dyed with BG1 and BG4, the comparison of two-dimensional spectra via principal component analysis (PCA) provided accurate fiber identification for all the analyzed fibers. The same approach was later applied to the investigation of laundering effects on the comparison of textile fibers. The presence of brighteners and other detergent components adsorbed in the fibers provided spectral fingerprints that enhanced the fiber identification process.The full dimensionality of EEMs was then explored with the aid of parallel factor analysis (PARAFAC), a second order algorithm capable to determine the number of fluorescence components that contribute to an EEM along with their individual excitation and emission profiles. The application of PARAFAC was carried out unsupervised and supervised by linear discrimination analysis (LDA). The classification performances of PARAFAC and LDA-supervised PARAFAC were compared to the one obtained with supervised discriminant unfolded partial least squares (DU-PLS). The best discrimination was obtained with the supervised DU-PLS, which allowed the pairwise differentiation of the four pairs of investigated fibers.DU-PLS was then used to investigate weathering effects on the spectral features of cotton 400 pre-dyed with DB1, nylon 361 pre-dyed with AY17 and acrylic 864 pre-dyed with BG4. The investigated fibers were exposed to humid (Florida) and dry (Arizona) weathering conditions for three, six, nine and twelve months. In all cases, this algorithm was unable to differentiate non-exposed acrylic fibers from exposed acrylic fibers. DU-PLS was able to differentiate non-exposed cotton and nylon fibers from exposed fibers to Florida and Arizona weathering conditions. It was possible to determine the period of exposure to either Florida or Arizona conditions. It was also possible to discriminate between fibers exposed to Florida or Arizona weathering conditions for the same period of time. These results provide the foundation for future studies towards a non-destructive approach capable to provide information on the history of the fiber.
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Date Issued
2016
Identifier
CFE0006838, ucf:51773
Format
Document (PDF)
PURL
http://purl.flvc.org/ucf/fd/CFE0006838
SYNTHESIS, CHARACTERIZATION, AND EVALUATION OF NEW REACTIVE TWO-PHOTON ABSORBING DYES FOR TWO-PHOTON EXCITED FLUORESCENCE IMAGING APPLICATIONS
Title
SYNTHESIS, CHARACTERIZATION, AND EVALUATION OF NEW REACTIVE TWO-PHOTON ABSORBING DYES FOR TWO-PHOTON EXCITED FLUORESCENCE IMAGING APPLICATIONS.
Creator
Hales, Katherine, Belfield, Kevin, University of Central Florida
Abstract / Description
Recent, cooperative advances in chemistry, computing, optics, and microelectronics have resulted in extraordinary developments in the biological sciences, resulting in the emergence of a novel area termed ¡¥biophotonics¡¦. The interdisciplinary nature of biophotonics cuts across virtually all disciplines, extending the frontiers of basic cellular, molecular, and biology research. This holds true for the development and application of the novel imaging modality utilizing...
Show moreRecent, cooperative advances in chemistry, computing, optics, and microelectronics have resulted in extraordinary developments in the biological sciences, resulting in the emergence of a novel area termed ¡¥biophotonics¡¦. The interdisciplinary nature of biophotonics cuts across virtually all disciplines, extending the frontiers of basic cellular, molecular, and biology research. This holds true for the development and application of the novel imaging modality utilizing multiphoton absorption and its extraordinary contribution to advances in bioimaging. Intimately involved in the revolution of nonlinear bioimaging has been the development of optical probes for probing biological function and activity. The focus of this dissertation is in the area of probe development, particularly ƒà-conjugated organic probes, optimized for efficient two-photon absorption followed by upconverted fluorescence for multiphoton bioimaging. Specifically, fluorene molecules, with enhanced two-photon absorbing (2PA) properties and high photostability, were prepared and characterized. Contemporary synthetic methods were utilized to prepare target fluorene derivatives expected to be highly fluorescent and, in particular, exhibit high two-photon absorptivity, suitable for two-photon excitation (2PE) fluorescence microscopy. The flexibility afforded through synthetic manipulation for integrating hydrophilic moieties into the fluorophore architecture to enhance compatibility with aqueous systems, more native to biological samples, was attempted. Incorporation of functional groups for direct covalent attachment onto biomolecules was also pursued to prepare fluorene derivatives as efficient 2PA reactive probes. Linear and two-photon spectroscopic characterizations on these novel compounds reveal they exhibit relatively high 2PA cross-sections on the order of ~100 GM units, which is greater than typical, commonly used fluorophores utilized in multiphoton bioimaging. Extensive photostability studies of representative fluorene compounds demonstrate these derivatives are photostable under one- and two-photon excitation conditions, exhibiting photodecomposition quantum yields on the order of 10-5. Additionally, preliminary cytotoxicity studies indicate these fluorene derivatives exhibit minimal cytotoxic effects on proliferating cells. Finally, their utility as high-performance, 2PA fluorescent probes in 2PE fluorescence microscopy imaging of biological samples was demonstrated in both fixed and live cells. Due to the low cytotoxicity, high photostability, efficient 2PA, and high fluorescence quantum yield, the probes were found suitable for relatively long-term, two-photon fluorescence imaging of live cells, representing a significant advance in biophotonics.
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Date Issued
2005
Identifier
CFE0000685, ucf:46487
Format
Document (PDF)
PURL
http://purl.flvc.org/ucf/fd/CFE0000685
SQUARAINE DYES FOR TWO-PHOTON FLUORESCENCE BIOIMAGING APPLICATIONS
Title
SQUARAINE DYES FOR TWO-PHOTON FLUORESCENCE BIOIMAGING APPLICATIONS.
Creator
Colon Gomez, Maria, Belfield, Kevin, University of Central Florida
Abstract / Description
Near-infrared emitting squaraine dyes are promising candidates for bioimaging applications. Two-photon fluorescence microscopy (2PFM) imaging is a powerful tool being used for studying biological function since it produces 3D images with minimal damage to cells and lower fluorophore photobleaching. The fluorescence wavelength of squaraine dyes normally falls in the near infrared region, providing deeper penetration through biological samples such as thick tissue sections. Squaraine dyes that...
Show moreNear-infrared emitting squaraine dyes are promising candidates for bioimaging applications. Two-photon fluorescence microscopy (2PFM) imaging is a powerful tool being used for studying biological function since it produces 3D images with minimal damage to cells and lower fluorophore photobleaching. The fluorescence wavelength of squaraine dyes normally falls in the near infrared region, providing deeper penetration through biological samples such as thick tissue sections. Squaraine dyes that could work for imaging cells and tissues for 2PFM imaging were synthesized and underwent comprehensive photophysical characterization, such as UV-Vis absorption, fluorescence, and anisotropy. The squaraine dyes were tested for cell toxicity to determine the concentration at which the cells should be incubated with the dye for 2PFM. In addition, the squaraine dyes were incubated with cancer cells to evaluate their utility in the bioimaging process. The squaraine dye that is not soluble in water can be incorporated in silica nanoparticles or micelles to facilitate dispersal in water for evaluation of its use as a probe. The prospective squaraine dyes can be used in cells and tissues for imaging that can then be analyzed to ascertain its use as a probe for biomedical applications, such as early cancer detection.
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Date Issued
2013
Identifier
CFH0004338, ucf:45020
Format
Document (PDF)
PURL
http://purl.flvc.org/ucf/fd/CFH0004338
Investigating New Guaiazulenes and Diketopyrropyrroles for Photonic Applications
Title
Investigating New Guaiazulenes and Diketopyrropyrroles for Photonic Applications.
Creator
Ghazvini Zadeh, Ebrahim, Belfield, Kevin, Campiglia, Andres, Yuan, Yu, Zou, Shengli, Cheng, Zixi, University of Central Florida
Abstract / Description
?-Conjugated systems have been the focus of study in recent years in order to understand their charge transport and optical properties for use in organic electronic devices, fluorescence bioimaging, sensors, and 3D optical data storage (ODS), among others. As a result, several molecular building blocks have been designed, allowing new frontiers to be realized. While various successful building blocks have been fine-tuned at both the electronic and molecular structure level to provide advanced...
Show more?-Conjugated systems have been the focus of study in recent years in order to understand their charge transport and optical properties for use in organic electronic devices, fluorescence bioimaging, sensors, and 3D optical data storage (ODS), among others. As a result, several molecular building blocks have been designed, allowing new frontiers to be realized. While various successful building blocks have been fine-tuned at both the electronic and molecular structure level to provide advanced photophysical and optoelectronic characteristics, the azulene framework has been under-appreciated despite its unique electronic and optical properties. Among several attributes, azulenes are vibrant blue naturally occurring hydrocarbons that exhibit large dipolar character, coupled with stimuli-responsive behavior in acidic environments. Additionally, the non-toxic nature and the accompanying eco-friendly feature of some azulenes, namely guaiazulene, may set the stage to further explore a more (")green(") route towards photonic and conductive materials.The first part of this dissertation focuses on exploiting guaiazulene as a natural building block for the synthesis of chromophores with varying stimuli-responsiveness. Results described in Chapter 1 show that extending the conjugation of guaiazulene through its seven-membered ring methyl group with aromatic substituents dramatically impacts the optical properties of the guaiazulenium carbocation. Study of these ?(-)stabilized tropilium ions enabled establishing photophysical structure-property trends for guaiazulene-terminated ?-conjugated analogs under acidic conditions, including absorption, emission, quantum yield, and optical band gap patterns. These results were exploited in the design of a photosensitive polymeric system with potential application in the field of three dimensional (3D) optical data storage (ODS).Chapter 2 describes the use of guaiazulene reactive sites (C-3 and C-4 methyl group) to generate a series of cyclopenta[ef]heptalenes that exhibit strong stimuli-responsive behavior. The approach presents a versatile route that allows for various substrates to be incorporated into the resulting cyclopenta[ef]heptalenes, especially after optimization that led to devising a one-pot reaction toward such tricyclic systems. Examining the UV-vis absorption profiles in neutral and acidic media showed that the extension of conjugation at C(4) of the cyclopenta[ef]heptalene skeleton results in longer absorption maxima and smaller optical energy gaps. Additionally, it was concluded that these systems act as sensitizers of a UV-activated ((<) 300 nm) photoacid generator (PAG), via intermolecular photoinduced electron transfer (PeT), upon which the PAG undergoes photodecomposition resulting in the generation of acid.In a related study, the guaiazulene methyl group at C-4 was employed to study the linear and nonlinear optical properties of 4-styrylguaiazulenes, having the same ?(-)donor with varying ?-spacer. It was realized that the conjugation length correlates with the extent of bathochromic shift of the protonated species. On the other hand, a trend of decreasing quantum yield was established for this set of 4-styrylguaiazulenes, which can be explained by the increasingly higher degree of flexibility.The second part of this dissertation presents a comprehensive investigation of the linear photophysical, photochemical, and nonlinear optical properties of diketopyrrolopyrrole (DPP)-based derivatives, including two-photon absorption (2PA), femtosecond transient absorption, stimulated emission spectroscopy, and superfluorescence phenomena. The synthetic feasibility, ease of modification, outstanding robustness, and attractive spectroscopic properties of DPPs have motivated their study for fluorescence microscopy applications, concluding that the prepared DPP's are potentially suitable chromophores for high resolution stimulated emission depletion (STED) microscopy.
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Date Issued
2015
Identifier
CFE0006034, ucf:50986
Format
Document (PDF)
PURL
http://purl.flvc.org/ucf/fd/CFE0006034
THE APPLICATION OF TWO-PHOTON ABSORBING PROBES IN LYSOSOMAL, ZINC ION SENSING AND FOLATE RECEPTOR-TARGETED BIOIMAGING
Title
THE APPLICATION OF TWO-PHOTON ABSORBING PROBES IN LYSOSOMAL, ZINC ION SENSING AND FOLATE RECEPTOR-TARGETED BIOIMAGING.
Creator
WANG, XUHUA, Belfield, Kevin D., University of Central Florida
Abstract / Description
Two-photon fluorescence microscopy (2PFM) has become a powerful technique for bioimaging in non-invasive cancer diagnosis and also investigating the mechanization and original of a variety of diseases by tracking various biological processes. Because the fluorescence emission by two photon absorbing (2PA) is directly proportional to the square of the intensity of excitation light, this intrinsic property of 2PA provides 2PFM great advantages over traditional one-photon fluorescence microscopy...
Show moreTwo-photon fluorescence microscopy (2PFM) has become a powerful technique for bioimaging in non-invasive cancer diagnosis and also investigating the mechanization and original of a variety of diseases by tracking various biological processes. Because the fluorescence emission by two photon absorbing (2PA) is directly proportional to the square of the intensity of excitation light, this intrinsic property of 2PA provides 2PFM great advantages over traditional one-photon fluorescence microscopy (1PFM), including high 3D spatial localization, less photodamage and interference from biological tissue because of using longer wavelength excitation (700-1300 nm). However, most 2PA probes are hydrophobic and their photostabilities are questionable, severely limiting their biological and medical applications. In addition, probes with significant specificity for certain organelles for tracking cellular processes or metal ions for monitoring neural transmission are somewhat rare. Moreover, it is also very significant to deliver the probes to specific disease sites for early cancer diagnosis. In order to increase the water solubility of probes, polyethylene glycol (PEG) was introduced to a fluorene-based 2PA probe LT1 for lysosomal 2PFM cell imaging. The 2PFM bioimaging application of the novel two-photon absorbing fluorene derivative LT1, selective for the lysosomes of HCT 116 cancer cells is described in Chapter II. Linear and nonlinear photophysical and photochemical properties of the probe were investigated to evaluate the potential of the probe for 2PFM lysosomal imaging. After the investigation of the cytotoxicity of this new probe, colocalization studies of the probe with commercial lysosomal probe Lysotracker Red in HCT 116 cells were conducted. A high colocalization coefficient (0.96) was achieved and demonstrated the specific localization of the probe in lysosomes. A figure of merit, FM, was introduced by which all fluorescent probes for 2PFM can be compared. LT1 was demonstrated to have a number of properties that far exceed those of commercial lysotracker probes, including much higher 2PA cross sections, good fluorescence quantum yield, and, importantly, high photostability, all resulting in a superior figure of merit. Consequently, 2PFM was used to demonstrate lysosomal tracking with LT1. In addition to lysosomes, it is also very significant to investigate the physiological roles of free metal ions in biological processes, especially Zn2+, because Zn2+ normally serves either as the catalytic elements in enzymatic activity centers or as structural elements in enzymes and transcription factors. However, biocompatible and effective Zn2+ probes for 2PFM bioimaging are infrequent. In Chapter III, 2PFM bioimaging with a hydrophilic 2PA Zn2+ sensing fluorescent probe, bis(1,2,3-triazolyl)fluorene derivative, is described. 2PFM bioimaging of the probe in living HeLa cancer cells was demonstrated. The results revealed a significant fluorescence increase upon introduction of Zn2+ into the cancer cells, and a reversible Zn2+ binding to the probe was also demonstrated, providing a robust probe for two-photon fluorescence zinc ion sensing. Early cancer diagnosis is another critical application for 2PFM, but there are still huge challenges for this new technique in clinical areas. Most 2PA probes with large two-photon absorbing cross sections and fluorescence quantum efficiency are synthetically more accessible in hydrophobic forms. In order to increase the efficiency of the probes and minimize the effect of the probe on the human body, delivery of the probe specifically to cancer sites is desired. The synthesis and characterization of narrow dispersity organically modified silica nanoparticles (SiNPs), diameter ~30 nm, entrapping a hydrophobic two-photon absorbing fluorenyl dye, are reported in Chapter IV. The surface of the SiNPs was functionalized with folic acid to specifically deliver the probe to folate receptor (FR) over-expressing HeLa cells, making these folate 2PA dye-doped SiNPs potential candidates as probes for two-photon fluorescence microscopy (2PFM) bioimaging. In vitro studies using FR over-expressing HeLa cells demonstrated specific cellular uptake of the functionalized nanoparticles. However, when the concentration of the dye in SiNPs increased for higher signal output, the fluorescence quantum efficiency of a probe normally decreases because of self-quenching. In Chapter V, a near-infrared (NIR) emitting probe is reported to overcome this limitation through both aggregate-enhanced fluorescence emission and aggregate enhanced two-photon absorption. The dye was encapsulated in SiNPs and the surface of the nanoparticles was functionalized with PEG followed by a folic acid derivative to specifically target folate receptors. NIR emission is important for deep tissue imaging. In vitro studies using HeLa cells that upregulate folate receptors indicated specific cellular uptake of the folic acid functionalized SiNP nanoprobe. Meanwhile, the probe was also investigated for live animal imaging by employing mice bearing HeLa tumors for in vivo studies. Ex vivo 2PFM tumor imaging was then conducted to achieve high quality 3D thick tissue tumor images.
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Date Issued
2011
Identifier
CFE0003640, ucf:48891
Format
Document (PDF)
PURL
http://purl.flvc.org/ucf/fd/CFE0003640