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Time Resolved Flourescence and Diffuse Reflectance Measurements for Lung Squamous Carcinoma Tumor Margins / OPTICAL PROPERTIES FOR LUNG CANCER MARGIN DETECTIONCosta, Sarah January 2023 (has links)
Lung cancer is the leading cause of death from cancer in Canada and is typically treated with surgical resection of the tumor. To ensure good prognosis and limit metastases no cancer cells can be left behind during resection. This project uses time-resolved fluorescence and diffuse reflectance to differentiate cancerous and non-cancerous lung tissue. These differences could be used during surgical resection of tumor to ensure no positive margins are present. Using a bi-modal spectroscopy device, BEAR, optical properties were determined for 36 tumor, 36 fibrotic and 9 normal lung tissue samples. Most optical parameters showed statistically significant differences between tumor and other tissue types. Metabolic based optical parameters showed statistically significant differences between fibrotic and normal tissue while non-metabolic based parameters showed no difference. As surgical margins are likely to be between tumor and fibrotic tissue the results demonstrate success and promise for implementing this system. Future work using fresh samples would develop the system further and would be a step closer to in vivo use during surgery. / Thesis / Master of Science (MSc) / Lung cancer is the leading cause of death from cancer and is typically treated by surgically removing the tumor. To improve survival all cancer cells must be removed which can be challenging. This project uses light to extract properties that can differentiate cancerous and non-cancerous lung tissue. These differences could be used during surgery to ensure no cancer cells remain. The project tests this system on 36 tumor, 36 fibrotic and 9 normal lung tissue samples. Most parameters showed significant differences between tumor and other tissue types. Given that often times the surgical boundaries are between tumor and fibrotic tissue the results demonstrate promise in implementing this system. Future work using fresh samples would develop the system further and bring it one step closer to being used during surgery.
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Targeting Melanocortin and Cholecystokinin Receptors via Multivalent Molecules Bearing Peptide LigandsNakath Gamlath Ralalage, Dayan Elshan January 2014 (has links)
Peptide receptor overexpression in diseased cells and tissues, including carcinomas provides an opportunity to develop therapeutics and imaging agents that selectively bind to such cells and tissues. This dissertation presents tools and processes that can be utilized to target melanocortin and cholecystokinin receptors through multivalent binding. In Chapter 2, improved synthesis and purification methods are described for the production of Eu-chelated probes that serve to evaluate the binding efficacy of multivalent molecules through competition binding assays. Specifically, a xylenol orange-based assay for quantification of unchelated metal ions was used to determine unbound metal ion contamination and the success of metal ion removal. The use of Empore™ chelating disks was determined to be the method of choice for the selective removal of unchelated Eu ions from several Eu-diethylenetriaminepentaacetic acid chelate-peptide conjugates. Applying new synthesis and purification strategies, the TRF probe Eu-DTPA-PEGO-CCK4 targeted to cholecystokinin receptors was synthesized (Chapter 2) and validated via saturation and competition binding assays (Chapter 4) using a HEK293 cell line overexpressing the human cholecystokinin 2 receptor. In Chapter 3, short and efficient syntheses of multivalent molecules targeted to melanocortin receptors based on three commercially available trigonal core scaffolds, phloroglucinol, tripropargylamine, and 1,4,7-triazacyclononane, are described. These constructs were designed to further test the 24 ±5 Å inter-ligand distance suggested in recent literature for multivalent binding to melanocortin receptors. The bioactivities of these compounds were evaluated using a competitive binding assay that employed HEK293 cells engineered to overexpress the human melanocortin 4 receptor. In the course of conducting these bioassays, novel in vitro binding assay protocols were established, which led to high repeatability and robustness of the bioassays compared to previous methods. The divalent molecules exhibited 10- to 30-fold higher levels of inhibition when compared to the corresponding monovalent molecules, consistent with divalent binding. The trivalent molecules were only statistically (~2-fold) better than the divalent molecules, still consistent with divalent binding but inconsistent with trivalent binding. Possible reasons for these behaviors and planned refinements of the multivalent constructs targeting melanocortin receptors based on these scaffolds are discussed in Chapters 3 and 6.
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A Diffusion Theory Model Of Spatially Resolved Fluorescence from Depth Dependent Fluorophore ConcentrationsHyde, Derek E. 09 1900 (has links)
Photodynamic therapy (PDT) currently utilizes drug and light doses which are primarily based on clinical experience. This can lead to a dose which is not sufficient to destroy the entire tumor, or alternatively, it can lead to the undesirable destruction of healthy tissue around the treatment area. PDT of topically applied photosensitizers is one focus of this research. This concerns the diffusion of an externally applied drug into the tissue, as well as its subsequent destruction during the irradiation procedure. This work involves the non-invasive measurement of the inherent fluorescence of the photosensitizer, allowing the determination of the concentration and distribution of drug within the tissue, and thus optimizing this treatment. To do this, one must be able to describe the propagation of light within the tissue. Consequently, a photon diffusion model has been developed to calculate the steady-state spatially resolved fluorescence from a pencil beam excitation in a depth dependent medium. The validity of this model was then verified by comparison with Monte Carlo simulations and measurements made on phantoms with optical properties similar to those of human tissue. Theoretical conditions were then explored, and potential uses of the model were demonstrated. / Thesis / Master of Science (MS)
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Use of green fluorescent protein for the analysis of protein-protein and protein-DNA interactionsChen, Kai January 2011 (has links)
Restriction modification (RM) systems play a crucial role in preventing the entry of foreign DNA into the bacterial cell. The best studied Type I RM system is EcoKI from Escherichia coli K12. Both bacteriophage and conjugative plasmids have developed a variety of strategies to circumvent the host RM system. One such strategy involves the production of antirestriction proteins that mimic a short segment of DNA and efficiently inhibit the RM system. The main aim of this project was to analyse the interaction of EcoKI and its cognate methylase (MTase) with the T7 antirestriction protein, known as overcome classical restriction (Ocr), and various ArdA antirestriction proteins. Currently, there is a paucity of structural data on the complex formed between the Type I system and the antirestriction proteins. The aim of this work was twofold; (i) compare the interaction of MTase with DNA and Ocr and (ii) quantify the strength of interaction between MTase and various ArdA proteins. The MTase was fused to the Green Fluorescent Protein (GFP) to facilitate determination of the orientation of interaction with DNA and Ocr. Time resolved fluorescence measurements were carried out using the GFP-MTase fusion to determine the fluorescence lifetime and anisotropy decay. These experiments were conducted using a time resolved fluorescence instrument fabricated in-house. The values determined in these experiments were then used to perform fluorescence resonance energy transfer (FRET) measurements with fluorescently labelled DNA or Ocr. These measurements gave information concerning the relative orientation of the MTase with either DNA or Ocr. The GFP-MTase fusion was also used to quantify the strength of interaction with various ArdA proteins. Previous attempts to determine the strength of interaction between MTase and ArdA proteins by employing conventional techniques have been unsuccessful. Therefore, a novel method was developed that exploits the interaction of MTase with a cation exchange medium, which can subsequently be displaced upon binding to ArdA. This method facilitated the determination, for the first time, of a set of binding affinities for the MTase and ArdA interaction.
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Development of a Time Resolved Fluorescence Spectroscopy System for Near Real-Time Clinical Diagnostic ApplicationsTrivedi, Chintan A. 2009 May 1900 (has links)
The design and development of a versatile time resolved fluorescence
spectroscopy (TRFS) system capable of near real time data acquisition and processing for
potential clinical diagnostic applications is reported. The TRFS apparatus is portable,
versatile and compatible with the clinical environment. The main excitation source is a
UV nitrogen laser with a nanosecond pulse width and the detection part consists of a dual
grating spectrograph coupled with an MCP-PMT. The nitrogen laser also has a dye
module attached to it, which enables broadband excitation of the sample. This setup
allows rapid acquisition (250 ms for fluorescence decay at a wavelength) of time resolved
fluorescence data with a high spectral (as low as 0.5 nm) and temporal (as low as 25
picoseconds) resolution. Alternatively, a state diode pumped pulsed laser can be used for
excitation to improve data collection speed. The TRFS system is capable of measuring a
broad range of fluorescence emission spectra (visible to near infra-red) and resolving a
broad range of lifetimes (ranging from a few hundred picoseconds to several
microseconds). The optical setup of the system is flexible permitting the connection of
different light sources as well as optical fiber based probes for light delivery/collection
depending on the need of the application. This permits the use of the TRFS apparatus in in vitro, ex vivo and in vivo applications. The system is fully automated for real-time data
acquisition and processing, facilitating near-real time clinical diagnostic applications.
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Automation of the Laguerre Expansion Technique for Analysis of Time-resolved Fluorescence Spectroscopy DataDabir, Aditi Sandeep 2009 December 1900 (has links)
Time-resolved fluorescence spectroscopy (TRFS) is a powerful analytical tool for quantifying the biochemical composition of organic and inorganic materials. The potentials of TRFS as nondestructive clinical tool for tissue diagnosis have been recently demonstrated. To facilitate the translation of TRFS technology to the clinical arena, algorithms for online TRFS data analysis are of great need.
A fast model-free TRFS deconvolution algorithm based on the Laguerre expansion method has been previously introduced, demonstrating faster performance than standard multiexponential methods, and the ability to estimate complex fluorescence decay without any a-priori assumption of its functional form. One limitation of this method, however, was the need to select, a priori, the Laguerre parameter a and the expansion order, which are crucial for accurate estimation of the fluorescence decay.
In this thesis, a new implementation of the Laguerre deconvolution method is introduced, in which a nonlinear least-square optimization of the Laguerre parameter is performed, and the selection of optimal expansion order is attained based on a Minimum Description Length (MDL) criterion. In addition, estimation of the zero-time delay between the recorded instrument response and fluorescence decay is also performed based on a normalized means square error criterion.
The method was fully validated on fluorescence lifetime, endogenous tissue fluorophores, and human tissue. The automated Laguerre deconvolution method is expected to facilitate online applications of TRFS, such as clinical real-time tissue diagnosis.
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Φασματοσκοπία χρονικής ανάλυσης και διφωτονικής απορρόφησης οργανικών ενώσεων παράγωγων της βενζοδισθιαζόληςΚοτσιάς, Δημήτριος 26 April 2012 (has links)
Στην παρούσα μεταπτυχιακή αυτή εργασία μελετήσαμε την συμπεριφορά για πρώτη φορά ενώσεων που είχαν σαν βάση την βενζοδισθιαζόλη. Συγκεκριμένα οι ενώσεις αυτές μελετήθηκαν με την χρήση των τεχνικών της φασματοσκοπίας διφωτονικής απορρόφησης της φασματοσκοπίας σταθερής κατάστασης και της φασματοσκοπίας φθορισμού χρονικής ανάλυσης.
Αρχικά όσον αφορά την φασματοσκοπία διφωτονικής απορρόφησης, μπορέσαμε να οδηγηθούμε στα εξής συμπεράσματα: οι καλύτερες ενώσεις που παρουσιάζουν αρκετά μεγάλη διφωτονική απορρόφηση είναι τα γραμμικά μόρια (PK-439 και PK-452) σε σχέση με τα U-shaped μόρια με μέγιστη ενεργό διατομή ΔΦΑ ~2000GM. Επιπλέον παρατηρήσαμε ότι η χρήση της βενζοδισθιαζόλης σαν κεντρικός πυρήνας προκαλεί σημαντική αύξηση της διφωτονικής απορρόφησης, σε σχέση με την βενζοθιαζόλη.
Τέλος, με την τεχνική της φασματοσκοπίας φθορισμού χρονικής ανάλυσης μπορέσαμε να οδηγηθούμε σε κάποιες διαπιστώσεις: συγκεκριμένα παρατηρήσαμε ότι όσο το μήκος κύματος καταγραφής μεγαλώνει, τόσο οι καμπύλες αποδιέγερσης γίνονται πιο αργές. Ακόμα διαπιστώσαμε ότι από την σύγκριση μορίων στο μήκος κύματος του μεγίστου, σε εκείνα τα μόρια που αποδιεγείρονται γρήγορα, ευνοούνται οι μη-ακτινοβολητικές διεργασίες και ταυτόχρονα παρουσιάζουν μικρή ενεργό διατομή ΔΦΑ. / --
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Integration of time-resolved fluorescence and diffuse reflectance spectroscopy for intraoperative detection of brain tumour marginnie, zhaojun 04 1900 (has links)
<p>The annual incidence rate of tumours in the brain and central nervous system (CNS) was 19.89 per 100,000 persons between 2004 and 2008 in the United States. Surgery is a common treatment option for brain and CNS tumours. Typically, biopsy followed by histological analysis is used to confirm tumour types and margin during neurosurgery as an intraoperative diagnostic tool. However, this biopsy method is invasive, sampling number limited and not in real-time. To overcome these problems, many minimally invasive optical techniques, called optical biopsies, have been developed towards intraoperative diagnosis.</p> <p>The research work carried out in this dissertation focuses on combining the time-resolved fluorescence (TRF) and diffuse reflectance (DR) spectroscopy towards intraoperative tumour margin detection in neurosurgery. Combining these two modalities allows us to obtain additional contrast features, thus potentially improving the diagnostic accuracy. To achieve this goal, first, a clinically compatible integrated TRF-DR spectroscopy instrument was developed for <em>in vivo</em> brain tumour study. An acousto-optical-tunable-filter-based spectrometer was designed to acquire the time-resolved fluorescence signal. A dual-modality fibre optic probe was used to collect the TRF and DR signals in a small volume. The system’s capabilities of resolving fluorescence spectrum and lifetime, and optical properties were characterized and validated using tissue phantoms. Second, in order to retrieve the fluorescence impulse response function accurately from measured fluorescence signals, a robust Laguerre-based deconvolution method was optimized by using the constrained linear least squares fitting and high order Laguerre function basis. This optimized Laguerre-based deconvolution method overcomes the over-fitting problem introduced by low signal-to-noise ratio and complex fitting model. Third, an <em>ex vivo</em> clinical study of brain tumours was carried out using the TRF and DR spectroscopy. Fluorescence spectra and lifetime features were selected to classify various tumour types. The sensitivity and specificity of meningioma grade I differentiated from meningioma grade II are both 100%. Finally, in order to increase the measurement tissue volume and obtain imaging contrast features, a scanning-based hyperspectral fluorescence lifetime imaging system was developed. This setup can provide time-, space-, spectrum- resolved multi-dimensional images for tumour margin detection.</p> / Doctor of Philosophy (PhD)
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Transition Dipole Moment and Lifetime Study of Sodium Dimer and Lithium Dimer Electronic States via Autler-Townes and Resolved Fluorescence SpectroscopySANLI, AYDIN January 2017 (has links)
This dissertation consists of three major studies. The first study, described in Chapter 3, focuses on the experimental work we carried out; experimental study of the electronic transition dipole moment matrix elements (TDMM) for the and electronic transitions of the sodium dimer molecule. Here we obtained the electronic transition dipole moments through Autler-Townes and resolved fluorescence spectroscopy and compared them to the theory. The second study, described in Chapter 4, is on sodium dimer ion-pair states. In this work, we calculated the radiative lifetimes and electronic transition dipole moments between Na2 ion-pair states ( , , , ) and state. This study was published in 2015. The last study, described in Chapter 5, is the total lifetime (bound-bound plus bound-free) and transition dipole moment calculations of the ion-pair electronic states, , of the lithium dimer molecule. / Physics
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Communication moléculaire photo-ionique : les études ultrarapides de composés supramoléculaireBatat, Pinar 24 October 2011 (has links)
Des molécules ou assemblages moléculaires organiques (dérivés d’hémicyanine ou du BODIPY) ont été étudiés en solution par des méthodes optiques complémentaires : absorption stationnaire et fluorescence, absorption transitoire et fluorescence résolue en temps (échelle femtoseconde et picoseconde). Ces méthodes ont permis de caractériser différents processus tels que le transfert de charge intramoléculaire, le transfert d'énergie et le transfert d'électron photoinduit. Elles ont ainsi permis de démontrer l’intérêt de certains chromophores de type AzaBODIPY émettant dans le proche IR, dans des applications d’imagerie et de thérapie photodynamique. La photostabilité et l’absorption à deux photons ont également été étudiées dans le cas d’autres dérivés du BODIPY pouvant être appliqués à la détection d’espèces ioniques. Dans le cas des hémicyanines, des dérivés amphiphiles dotés d’une couronne reconnaissant spécifiquement certains cations ont également été étudiés sous forme de films de Langmuir-Blodgett et en présence de différents cations, le but étant de former des membranes artificielles iono- et photosensibles. / Ultrafast femtosecond transient absorption measurements (30 fs FWHM pulses) and complementary picosecond spectroscopies (20 ps FWHM pulses, streak camera detection), as well as steady state absorption and fluorescence measurements, were used to study a range of molecules and molecular assemblies. Processes such as intramolecular charge transfer, electronic energy transfer and photoinduced electron transfer were characterized. Amphiphilic azacrown-containing hemicyanine dyes and resulting iono- and photosensitive artificial membranes were studied using Langmuir-Blodgett techniques in the presence of various cations. Among a range of other molecules studied, NIR emitting aza-BODIPY dyes were studied by time-resolved methods in order to investigate their suitability for Photodynamic Therapy applications and imaging. Differently functionalized BODIPY dyes were investigated with respect to photostability, two photon absorption and ion sensing.
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