acase@tulane.edu / Current microscopy-based tissue diagnostics, particularly hematoxylin and eosin (H&E) histology, requires multiple complex tissue processing steps: fixation, paraffin embedding, microtome sectioning, dying the tissue, and imaging individual slides through a bright field microscope. The time and labor-intensive result of this process makes it unsuitable for patient point-of-care evaluation. Therefore, many bedside procedures are completed without efficient real-time analysis of tissue adequacy and diagnostic results are unnecessarily delayed. Additionally, research experiments that require information regarding changes to tissue morphology or function before proceeding to the next experimental phase are severely interrupted by histology processing in their workflow. Fluorescence histology, which relies on rapid fluorescent staining of tissue, optical sectioning microscopy, and image processing for digital viewing, can provide an inexpensive, non-destructive, 3-dimensional, and fast alternative to traditional histology and point-of-care screening protocols. The objective of this work is to further advance the concept of “fluorescence histology,” in which traditional histopathology preparation methods are replaced by optical-sectioning (in lieu of physical sectioning), sensitive and flexible fluorescence-based contrast (in lieu of chromophore-based contrast), and computational strategies to replicate traditional color-schemes. In this work, we demonstrate the development and use of a fluorescent analogue to H&E on fixed and frozen tissue sections and fresh human biopsies. This fluorescent analogue, DRAQ5 & eosin, is compared against the current single-agent, monochrome fluorescence histology system, and their effects on diagnostic downstream molecular analyses, including quantitative-PCR and immunohistochemistry, is evaluated. We create a methodology to develop and characterize fluorescent analogues for any histological stain, with demonstration using Masson’s Trichrome and Periodic Acid-Schiff, enabling the expansion of fluorescence histology for multiple applications. This work demonstrates the ability to improve point-of-care pathology and research by replacing destructive, incomplete, and time-consuming histology with fluorescence histology, which preserves the tissue for later analysis or experiments while providing accurate and rapid histology assessment. / 1 / Katherine Elfer

Bacteria are implicated in endodontic infections. They not only infect the root canal lumen but also invade the dentinal tubules where they may remain untouched by contemporary chemomechanical preparation during root canal therapy. The contentious issue is whether the bacteria within these tubules contribute to secondary infections. Many studies have shown that clinicians fail to completely eradicate them during root canal therapy. At present there are no techniques available to detect the effectiveness of the current chemomechanical treatment regime within dentinal tubules. It is difficult to detect bacteria within the dentinal tubules. Culturing techniques have been used routinely as they are versatile and easy to use. However, they are unable to show the distribution of the bacteria within the dentinal tubules. Scanning electron microscopy, on the other hand, shows detailed surface structure in association with bacteria. Histological examination of root dentine specimens under the light microscope also shows the distribution of bacteria within the specimen but not viability. The dilemma posed by these existing techniques is that the results offer limited information; either demonstrating bacterial viability or bacterial distribution within specimens. No techniques able to show both the viability and the distribution of bacteria within the dentinal tubules have been reported to date. Fluorescent stains, in particular SYTO�9 and propidium iodide (LIVE/DEAD� Baclight[TM] viability kit, Molecular Probes Inc., Eugene, Oregon), have made it possible not only to stain bacteria but to differentiate live and dead bacteria. The combination of these two stains has yet to be applied to dental hard tissue in situ and they provide the basis for this investigation. The aim of this study was to evaluate the potential of the LIVE/DEAD� Baclight[TM] stains in conjuction with confocal laser scanning microscopy in the development of a technique to evaluate the viability and distribution of bacteria within dentinal tubules. This was extended to demonstrate the application of this technique by examining three different means of root canal disinfection both qualitatively and quantitatively. An important aspect of this study was to maintain bacterial viability, as well as to get maximum bacterial invasion into dentinal tubules. Results indicated that when the root canals were instrumented with Protaper� files and then irrigated with sodium hypochlorite (NaOCl) and ethylene diaminetetraacetic acid with cetrimide (EDTAC), there was more bacterial invasion into the dentinal tubules than when the root canals were only irrigated with NaOCl and EDTAC. Daily replenishments of nutrients resulted in deeper bacterial invasion into the dentinal tubules. Bacteria colonized the dentinal tubules up to a distance of 594 � 133 [mu]m from the canal. In the untreated tubules, 96 � 4 % of bacteria remained viable (green-fluorescent), whereas the Amoxicillin-treated tubules contained 94 � 6 % dead (red-fluorescent) bacteria. The calcium hydroxide-treated tubules resulted in 92 � 7 % bacterial death while the laser-treated tubules contained 81 � 12 % dead cells, frequently displaying an inner zone of dead cells surrounded by an outer zone of viable cells. The application of the fluorescent stains combined with confocal microscopy offers a new method for assessing the in vitro efficacy of root canal disinfection regimens.

Thesis (M.S.)--University of Nevada, Reno, 2005. / "December, 2005." Includes bibliographical references (leaves 50-53). Online version available on the World Wide Web.

Thesis (Ph. D.)--West Virginia University, 2006. / Title from document title page. Document formatted into pages; contains xii, 146 p. : ill. (some col.). Includes abstract. Includes bibliographical references (p. 134-146).

Thesis (Ph. D.)--University of Washington, 2003. / Vita. Includes bibliographical references (leaves 156-160).

This research focuses on developing selective FRET peptidyl metal ion sensors as a portable and less costly alterative to traditional atomic spectrometric techniques. Initially, a selective sensor for Cu²⁺ was developed that consisted of glycine and aspartic acid residues and the FRET pair tryptophan (donor) and dansyl (acceptor). Aspartic acid's affinity for hard acid metals and Cu²⁺'s preference for square planar coordination was used as the basis of design. Although the sensor was designed to utilize the signal enhancement capabilities of FRET, quenching of both fluorophores occurred and proved to be the most sensitive means of quantifying Cu²⁺ binding. Nonetheless, the sensor provided a selective and sensitive response to Cu²⁺ at pH 7.0. Another FRET peptide metal ion sensor was designed with the help of a biological starting point, the mercury binding protein MerP. A sensitive FRET enhancement or "turn on" response was observed for Hg²⁺, as well as Zn²⁺, Cd²⁺ and Ag²⁺ in pH 7.0 solution. While a selective response for only Hg²⁺ was the ultimate goal of this study, this sensor is still an improvement over current systems which utilize a quenching mechanism for Hg²⁺ detection. While the previous studies investigated these sensors in aqueous solutions, the end goal was to devise a sensor based on an immobilized peptide chelator with FRET capabilities. To this end, immobilized, fluorophore labeled peptide studies were then conducted on Tentagel resin using a visible region FRET pair. A flow injection fluorescence analysis system using the immobilized fluorophore labeled peptide as the ion exchange material was also designed, allowing for the efficient analysis of fluorescence solutions. In addition to the work conducted with FRET sensors, studies were also conducted using magnetic [gamma]-Fe₂O₃ nanoparticles with PLCys immobilized onto the surface. The [gamma]-Fe₂O₃ nanoparticles are ideal supports since they can be magnetically collected and have a very large surface area to mass ratio. Finally, a method was developed to quantitatively screen metals bound to single Tentagel beads with immobilized peptides using ETV-ICP-MS. This method is an improvement over existing methods because it is nondestructive and simultaneously provides the absolute content of all metals bound.

The carcinoembryonic antigen-related cellular adhesion molecule 1 (CEACAM1) is a human receptor that facilitates adhesion with neighbouring cells, as well as with certain pathogens. CEACAM1 at the cell surface exists as a mixture of monomers and dimers in a heterogeneous distribution that is thought to regulate the balance of its functions, including those associated with pathogen binding. We used live cell fluorescence and homogeneous Förster resonance energy transfer (homo-FRET) microscopy on a combined total internal reflection fluorescence polarization (TIRFPM) confocal microscopy platform to investigate the distribution, dynamics, and monomer-dimer equilibrium of CEACAM1-4L-EYFP on live cells that were parachuted onto surfaces coated with CEACAM1-binding Neisseria gonorrhoea. Both CEACAM1-4L-EYFP and a monomeric mutant form of the receptor are rapidly recruited to bacteria and lead to downstream effector recruitment. Homo-FRET data indicate that wild-type CEACAM1-4L-EYFP was predominantly monomeric at bacterial contact sites. Preferential monomeric binding during bacterial adhesion controls the infection process.

The carcinoembryonic antigen-related cellular adhesion molecule 1 (CEACAM1) is a human receptor that facilitates adhesion with neighbouring cells, as well as with certain pathogens. CEACAM1 at the cell surface exists as a mixture of monomers and dimers in a heterogeneous distribution that is thought to regulate the balance of its functions, including those associated with pathogen binding. We used live cell fluorescence and homogeneous Förster resonance energy transfer (homo-FRET) microscopy on a combined total internal reflection fluorescence polarization (TIRFPM) confocal microscopy platform to investigate the distribution, dynamics, and monomer-dimer equilibrium of CEACAM1-4L-EYFP on live cells that were parachuted onto surfaces coated with CEACAM1-binding Neisseria gonorrhoea. Both CEACAM1-4L-EYFP and a monomeric mutant form of the receptor are rapidly recruited to bacteria and lead to downstream effector recruitment. Homo-FRET data indicate that wild-type CEACAM1-4L-EYFP was predominantly monomeric at bacterial contact sites. Preferential monomeric binding during bacterial adhesion controls the infection process.

Bacteria are implicated in endodontic infections. They not only infect the root canal lumen but also invade the dentinal tubules where they may remain untouched by contemporary chemomechanical preparation during root canal therapy. The contentious issue is whether the bacteria within these tubules contribute to secondary infections. Many studies have shown that clinicians fail to completely eradicate them during root canal therapy. At present there are no techniques available to detect the effectiveness of the current chemomechanical treatment regime within dentinal tubules. It is difficult to detect bacteria within the dentinal tubules. Culturing techniques have been used routinely as they are versatile and easy to use. However, they are unable to show the distribution of the bacteria within the dentinal tubules. Scanning electron microscopy, on the other hand, shows detailed surface structure in association with bacteria. Histological examination of root dentine specimens under the light microscope also shows the distribution of bacteria within the specimen but not viability. The dilemma posed by these existing techniques is that the results offer limited information; either demonstrating bacterial viability or bacterial distribution within specimens. No techniques able to show both the viability and the distribution of bacteria within the dentinal tubules have been reported to date. Fluorescent stains, in particular SYTO�9 and propidium iodide (LIVE/DEAD� Baclight[TM] viability kit, Molecular Probes Inc., Eugene, Oregon), have made it possible not only to stain bacteria but to differentiate live and dead bacteria. The combination of these two stains has yet to be applied to dental hard tissue in situ and they provide the basis for this investigation. The aim of this study was to evaluate the potential of the LIVE/DEAD� Baclight[TM] stains in conjuction with confocal laser scanning microscopy in the development of a technique to evaluate the viability and distribution of bacteria within dentinal tubules. This was extended to demonstrate the application of this technique by examining three different means of root canal disinfection both qualitatively and quantitatively. An important aspect of this study was to maintain bacterial viability, as well as to get maximum bacterial invasion into dentinal tubules. Results indicated that when the root canals were instrumented with Protaper� files and then irrigated with sodium hypochlorite (NaOCl) and ethylene diaminetetraacetic acid with cetrimide (EDTAC), there was more bacterial invasion into the dentinal tubules than when the root canals were only irrigated with NaOCl and EDTAC. Daily replenishments of nutrients resulted in deeper bacterial invasion into the dentinal tubules. Bacteria colonized the dentinal tubules up to a distance of 594 � 133 [mu]m from the canal. In the untreated tubules, 96 � 4 % of bacteria remained viable (green-fluorescent), whereas the Amoxicillin-treated tubules contained 94 � 6 % dead (red-fluorescent) bacteria. The calcium hydroxide-treated tubules resulted in 92 � 7 % bacterial death while the laser-treated tubules contained 81 � 12 % dead cells, frequently displaying an inner zone of dead cells surrounded by an outer zone of viable cells. The application of the fluorescent stains combined with confocal microscopy offers a new method for assessing the in vitro efficacy of root canal disinfection regimens.

Thesis (Ph. D.)--Missouri University of Science and Technology, 2009. / Vita. The entire thesis text is included in file. Title from title screen of thesis/dissertation PDF file (viewed April 29, 2009) Includes bibliographical references.