No description available.

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.

This project explores optimization of two-photon excited fluorescence (TPEF) enhancement between tunable narrowband and un-tuned broadband femtosecond (fs) laser pulse excitations for two-photon microscopy (TPM). The research is conducted preliminarily in time domain and comprehensively in frequency domain to understand the physics behind TPEF enhancement by un-tuned sub-10 fs nearly transform-limited pulse (TLP) versus tunable 140 fs pulse. The preliminary study on inverse proportionality of TPEF yield to fs-pulse duration delimits a general lower-bound to narrowband fs-pulse regime (pulse duration > 40 fs) with assumption of dye-molecule frequency invariant response. Deviations from this inverse proportionality in broadband fs-pulse regime (pulse duration < 40 fs) highlights dye-molecule frequency variant response, necessity of group delay dispersion (GDD) compensation, and broadband TLP for TPEF enhancement. The follow-up comparative study is made on un-tuned sub-10 fs TLP versus tunable 140 fs pulse excitations using three dye-phantoms (Indo-1, FITC, and TRITC) representative of fluorescent probes with similar TPEF characteristics. The integrated experimental system, with custom-designed GDD compensation, dispersion-less laser-beam expanding and focusing, and compound-lens for efficient fluorescence collection with good spectral resolution, ensures accurate TPEF measurements. Differentiated TPEF enhancements of Indo-1 (1.6), FITC (6.7), and TRITC (5.2) proportionally agree with calculated ones due to the overlap of fs-pulse second harmonic (SH) power spectrum with dye-molecule two-photon excitation (TPE) spectrum. Physically speaking, with broadband sub-10 fs TLP readily involved in both degenerate (v1 = v2) and non-degenerate (v1 ≠ v2) two-photon absorption (TPA), this un-tuned ultrashort fs-pulse excitation simultaneously allows for more accessibility to TPA-associated final states and diversely promotes population of thus excited dye-molecules with the three dye-phantoms. Under environmental influences (mutual quenching through one-photon absorption(s) and solvent effect), multicolor TPEF enhancement observed from a mixture of the three dyes shows promise of sub-10 fs TLP as simultaneous excitation for multiple-dye labeled samples in contrast to compromised excitation with narrowband fs-pulse tuning. Both single- and multicolor TPEF enhancements clarify tradeoff between tunability of narrowband fs-pulse and un-tuned broadband fs-pulse excitations, being instructive to further considerations on optimization of TPEF enhancement by strategic utilization of broadband fs-pulse for better performance of TPM.

Two families of amphiphilic polymers were characterized with respect to their ability to respond to changing solution conditions using steady-state and time-resolved fluorescence. Firstly, a series of 5 sequential amphiphilic polypeptides consisting of hydrophilic and ionizable aspartic acid (Asp) and hydrophobic phenylalanine (Phe) in varying sequence (AspxPhey)n were studied. The effect of pH on the collapse and aggregation behaviour of the samples was investigated using light scattering which determined that the samples became insoluble when the fraction of ionized amino acids decreased below 0.2. Pyrene fluorescence measurments demonstrated that hydrophobic aggregate formation was the cause of this behaviour. The fluorescence and light scattering experiments yielded a detailed description of how pH affects the collapse and aggregation behaviour of amphiphilic polypeptides. Secondly, a series of poly(N-isopropylacrylamide) chains in aqueous solution, varying in length and end-labelled with pyrene (Py2-PNIPAM), were studied with respect to their temperature dependent solubility using turbidimetry, light scattering, and fluorescence. The cloud point temperature (Tc) of the Py2-PNIPAM samples was found to increase with increasing chain length. Steady-state fluorescence spectra yielded the ratio of excimer intensity to that of monomer intensity, or (IE/IM)SS, which reached a maximum at Tc. Time-resolved fluorescence decays were analyzed using Model-Free Analysis (MFA). The MFA yielded the average rate constant of excimer formation <k>, and the parameters fagg, fdiff and ffree, which reflect the molar fractions of pyrenes that form excimer from pre-formed aggregates, form excimer by diffusion, and do not form excimer, respectively. Increasing the temperature above Tc caused a strong decrease in <k> and ffree, and a sharp rise in fdiff, which is consistent with the formation of mesoglobules. Increasing the temperature above the dehydration temperature of unlabelled PNIPAM (Tm = 34 oC) caused the distribution of pyrene to stabilize. The third study focused on aqueous mixtures of Py2-PNIPAM and unlabelled PNIPAM, and their ability to mix when the temperature was raised above Tm. Using turbidimetry, separate transitions for Py2-PNIPAM and unlabelled PNIPAM were identified. Steady-state fluorescence of the labelled chains demonstrated separate (IE/IM)SS transitions for the labelled and unlabelled chains as well. The MFA of the time-resolved fluorescence decays yielded no transition in <k> at Tm, while fagg, fdiff and ffree showed transitions consistent with unlabelled PNIPAM entering the Py2-PNIPAM mesoglobules. This led to the conclusion that the two polymers are able to mix at and above Tm, and that the mesoglobules are not frozen and vitreous. Fourthly, a variety of pyrene-labelled polymers in organic solution were studied using steady-state and time-resolved fluorescence, where the results of Birks’ Scheme and the Fluorescence Blob Model (FBM) were compared to the results of the MFA. The MFA was found to be able to faithfully reproduce the results of the other, more established, models. In addition, the MFA allowed the calculation of (IE/IM)SPC, the ratio of excimer to monomer intensity as determined by time-resolved fluorescence, which scaled linearly with both (IE/IM)SS and <k>. From this it was concluded that the MFA is able to fit the decays of pyrene-labelled polymers with any architecture studied thus far, but also provides an absolute measure of IE/IM which can be reproduced in any lab.

The quality of machinery liquids plays a critical role in ensuring safe and cost-effective operation of engines. Especially in the aviation industry, there is a great need for real-time and online monitoring of the purity, lubricity and age of machinery fluids. In this work, two optical techniques, excitation-emission matrix spectroscopy (EEMS) and cavity ring-down spectroscopy (CRDS), are used for monitoring of degradation and contamination of aero-turbine lubricants and jet fuels using optical fiber probes. We implement EEMS combined with a modified fiber probe design to characterize lubricant quality through the characteristic fluorescence of antioxidant additives. Multi-way analysis procedures, such as parallel factor analysis, are applied to correlate spectral features to antioxidant concentration, oxidative stability, and lubricant age. The spectroscopic data are then correlated to commonly used, off-line parameters such as the induction time and the breakdown number. It is shown that the decrease in fluorescence intensities of antioxidants coincides with the decomposition of the oil base stock. The induction times of synthetic jet turbine oil degraded at 150ºC, 195ºC and 215ºC are found to be at about 10,000, 3,500 and 400 min respectively. Simple kinetic models are developed that are capable of describing antioxidant reactions as pseudo first-order processes. We also demonstrate that with fluorescence detection it is possible to determine the concentration of oil contamination in jet fuel from about 10 to 1000 ppmv. In addition, a fiber-loop cavity ring-down spectrometer has been developed to quantitatively identify oil contamination of jet fuel by measuring optical absorption in the UV region. CRDS is a very sensitive, path-enhanced absorption technique that may be used for trace-species measurements in gas and liquids. The absorption measurements on samples with small volumes are characterized by measuring the concentration of turbine oil in jet fuel from 100 000 ppmv to a limit of detection of 400 ppmv. In summary, the obtained results permit us to specify the life time of lubrication oil and to determine the contamination of jet fuel with turbine oil qualitatively and quantitatively. In a simple optical configuration the fiber-coupled EEM and CRD methods permit in situ sampling of the machinery fluids. / Thesis (Ph.D, Chemistry) -- Queen's University, 2014-04-25 13:24:37.761

The repression of the E. coli lac operon seems to be more efficient than the current theoretical model allows for. Specifically, it is more quiet than expected during the replication of the chromosome. I have induced cells during short periods and counted the number of protein products from the operon to determine if there is a delay in activation of transcription that could account for the discrepancy. The results are compatible with a delay of 10-20 s, but the delay could not be conclusively proven. Furthermore, it has been investigated if the mechanism behind the delay might be differential localization of the lac operon with and without induction. It is shown that the lac operon is more often located in the periphery of the cell and in the internucleoid region when induced. These might be regions where genes are higher expressed, giving a delay in expression after de-repression before the gene is transported there.

No description available.

The unique optical properties of quantum dots (QDs) have been widely used to develop bioassays based on Fluorescence Resonance Energy Transfer (FRET). The solid-phase assays using QDs as FRET donors have numerous practical advantages, including at least 10-fold enhancement in FRET efficiency, which is not immediately explained by theoretical predictions that model energy transfer processes of QDs in two-dimensional layers. Donor-acceptor separation distance, acceptor and donor concentrations were found to influence FRET efficiency in solid-phase assays. A novel immobilization strategy was implemented which made use of the high affinity of imidazole moieties to QD shells to build solid-phase QD bioassays. A 96-well polystyrene plate is presented as a platform suitable for rapid and convenient multiplexed detection. A typical microtiter plate reader was shown to be capable of discriminating different FRET pairs to picomol detection levels of target oligonucleotides. Furthermore, the QD-FRET bioassays provided for mismatch discrimination, and multiple cycles of regeneration were also demonstrated.

The exploration of a quantum dot fluorescence resonance energy transfer (QD-FRET) based bioassay for label-free target nucleic acid detection is reported herein. This work explores the potential for developing a displacement assay for detection of nucleic acid sequences of various lengths, including one of 484 bases. Short probe oligonucleotides conjugated to QDs were allowed to hybridize to short partially mismatched dye-labelled oligonucleotide targets. The non-labelled target of interest, a 484-base segment of heat shock protein 70 (HSP 70), contained a portion that was fully complementary to the probe. Thermodynamic parameters suggested that HSP 70 would displace dye-labelled targets; however, detection was not observed. Modifications were made to this assay to reduce sterics and increase the stability of hybrids. The results obtained using this modified assay indicated that detection of non-labelled, long oligonucleotide sequences was possible using a displacement assay that relied on a short probe oligonucleotide.

The unique optical properties of quantum dots (QDs) have been widely used to develop bioassays based on Fluorescence Resonance Energy Transfer (FRET). The solid-phase assays using QDs as FRET donors have numerous practical advantages, including at least 10-fold enhancement in FRET efficiency, which is not immediately explained by theoretical predictions that model energy transfer processes of QDs in two-dimensional layers. Donor-acceptor separation distance, acceptor and donor concentrations were found to influence FRET efficiency in solid-phase assays. A novel immobilization strategy was implemented which made use of the high affinity of imidazole moieties to QD shells to build solid-phase QD bioassays. A 96-well polystyrene plate is presented as a platform suitable for rapid and convenient multiplexed detection. A typical microtiter plate reader was shown to be capable of discriminating different FRET pairs to picomol detection levels of target oligonucleotides. Furthermore, the QD-FRET bioassays provided for mismatch discrimination, and multiple cycles of regeneration were also demonstrated.