Academic literature on the topic 'Fluorescence spectroscopy'

Fluorescence correlation spectroscopy has become a standard technique for modern biophysics and single molecule spectroscopy research. Here is presented a novel widefield extension of the established single-point technique. Flow in microfluidic devices was used as a model system for microscopic motion and through widefield fluorescence correlation spectroscopy flow profiles were mapped in three dimensions. The technique presented is shown to be more tolerant to low signal strength, allowing image data with signal-to-noise values as low as 1.4 to produce accurate flow maps as well as utilizing dye-labeled single antibodies as flow tracers. With proper instrumentation flows along the axial direction can also be measured. Widefield fluorescence correlation spectroscopy has also been utilized to produce super-resolution confocal microscopic images relying on the single-molecule microsecond blinking dynamics of fluorescent silver clusters. A method for fluorescence modulation signal extraction as well as synthesis of several novel noble metal fluorophores is also presented.

Fluorimetry in the very near infrared region ca. 600-1000nm is a new approach to photochemical analysis. The advantages include greatly reduced background fluorescence signals from important sample matrices (such as blood serum), reduced scattering, and reduced probability of sample decomposition. Also, the availability of low cost, efficient, stable and robust optical components (e.g. laser diodes and light emitting diodes), solid state detectors (e.g. single silicon photodiodes and diode arrays) and fibre optics, allows the construction of an inexpensive fluorimeter. In the near infrared region, there are some very bright fluorophores that can be adapted for use as fluorescent probes, labels for immunoassay, and as ion-pair agents. The advantageous performance of most types of fluorimetric analysis now undertaken In the ultraviolet and visible region of the spectrum may therefore be extended into the longer wavelength region. Excellent limits of detection are attainable, and some near infrared fluorophores show invaluable fluorescence probe properties, such as Nile Red. The most useful of the dye groups investigated were the phenoxazines and thiazines. Reactive derivatives of these dyes show great potential as fluorescent labels for Immunoassay. These dyes have also been used as probes due to their solvatochromism and sensitivity to pH.

Corrected steady-state fluorescence emission spectra of various mechanical pulps and celluloses were measured using an excitation wavelength of 320 nm. Regardless of its source, cellulose displayed a relatively high characteristic emission. The emission spectra of mechanical pulps were red-shifted with respect to that of cellulose, and peak shapes were influenced by the pulping process, by hydrogen peroxide bleaching and by monochromatic UV irradiation of the sheets. Changes in emission spectra due to bleaching or UV-irradiation occurred principally in the 370-440 and 500-550 nm regions, and were similar to changes in reflectance spectra reported in the literature. Fluorescence emission spectra of commercial lignins and lignin model compounds were blue-shifted with respect to mechanical pulps, even upon absorption of the fluorophores on solid substrates. Concentration and inner filter effects were evident in emission spectra of commercial lignins, at concentrations as low as 60 ppm. These results suggest that, to a first approximation, emission spectra of mechanical pulps may be interpreted as a decrease in emission of cellulose caused by light absorption by lignin; changes due to bleaching and irradiation may be attributed to changes in lignin absorption.

During biosynthesis on the ribosome, the nascent polypeptide chain emerges in a vectorial fashion, one amino acid at a time and it has its first opportunity to sample conformational space. Study of structural and molecular biology has shown that nascent chains (NC) can co-translationally acquire its native structure, however how an emerging NC forms the native conformation is a fundamental question in modern biology. Yellow fluorescent protein (YFP), a red shifted yellow emission variant of green fluorescent protein (GFP) that is consisted of 238 amino acid residues is highly distinct because of its fast maturation of fluorophore and its rapid folding kinetics. In addition, YFP has been extensively studied using X-ray crystallography, nuclear magnetic resonance (NMR), and biophysics, which make it an ideal system for examining protein folding on the ribosome. In this study, therefore, we created a series of ribosome-nascent chain complex (RNC) of YFP to simulate the progressive emergence of NC. We also developed an in vitro transcription translation system to generate isotopically-labelled RNCs for both biophysics and NMR spectroscopy. To complement the RNC study, we also created a co-translational folding mimetic using C-terminal truncations of isolated YFP to evaluate folding in vitro. Using these approaches, the co-translational folding of YFP was observed using both spectroscopies of fluorescence and NMR at a residue specific level. Here we demonstrate that native folding can take place very close to the ribosome in which YFP is separated by 19 amino acids from the ribosome’s peptidyl transferase centre (PTC), and that YFP can tolerate the absence of up to 14 amino acids from its C-terminus and still acquire a native fold.

Protein based biopharmaceuticals are becoming increasingly popular therapeutic agents. Recent changes to the legislation governing stem cell technologies will allow many further developments in this field. Characterisation of these therapeutic proteins poses numerous analytical challenges. In this work we address several of the key characterisation problems; detecting glycosylation, monitoring conformational changes, and identifying contamination, using vibrational spectroscopy. Raman and infrared spectroscopies are ideal techniques for the in situ monitoring of bioprocesses as they are non-destructive, inexpensive, rapid and quantitative. We unequivocally demonstrate that Raman spectroscopy is capable of detecting glycosylation in three independent systems; ribonuclease (a model system), transferrin (a recombinant biopharmaceutical product), and GFP (a synthetically glycosylated system). Raman data, coupled with multivariate analysis, have allowed the discrimination of a glycoprotein and the equivalent protein, deglycosylated forms of the glycoprotein, and also different glycoforms of a glycoprotein. Further to this, through the use of PLSR, we have achieved quantification of glycosylation in a mixture of protein and glycoprotein. We have shown that the vibrational modes which are discriminatory in the monitoring of glycosylation are relatively consistent over the three systems investigated and that these bands always include vibrations assigned to structural changes in the protein, and sugar vibrations that are arising from the glycan component. The sensitivity of Raman bands arising from vibrations of the protein backbone to changes in conformation is evident throughout the work presented in this thesis. We used these vibrations, specifically in the amide I region, to monitor chemically induced protein unfolding. By comparing these results to fluorescence spectroscopy and other regions of the Raman spectrum we have shown that this new method provides improved sensitivity to small structural changes. Finally, FT-IR spectroscopy, in tandem with supervised machine learning methods, has been applied to the detection of protein based contaminants in biopharmaceutical products. We present a high throughput vibrational spectroscopic method which, when combined with appropriate chemometric modelling, is able to reliably classify pure proteins and proteins ‘spiked’ with a protein contaminant, in some cases at contaminant concentrations as low as 0.25%.

Ce projet de recherche conjugue deux aspects complémentaires : le développement d’un montage de microscopie intégrant un système d'Optique Adaptative (OA) et l’étude de masses cancéreuses (Sphéroïdes Multicellulaires) sous pression mécanique.Ces deux axes seront mutuellement bénéfiques puisque l’implémentation de l’OA rendra possible l’imagerie et les mesures physiques au sein des sphéroïdes ; d’un autre côté, l’étude des sphéroïdes permettra de caractériser les aberrations induites par ce type d’échantillons et de mieux comprendre les exigences sur le système d’OA qu’imposent l’observation de ces échantillons ainsi que les limites de la microscopie optique dans les tissus biologiques<br>This research project combines two complementary aspects: the development of an assembly incorporating an Adaptive Optics microscope system and the study of cancerous masses (multicellular spheroids) under mechanical pressure.These two axes are mutually beneficial since the implementation of the adaptive optics will enable imaging and physical measurements in spheroids; On the other hand, the study of spheroids will characterize the aberrations induced by this type of samples and understand the requirements of the adaptive optics system imposed by the observation of these samples as well as the limits of optical microscopy in biological tissues

This thesis describes the experimental investigations concerning the integration and optimisation of a supercontinuum source into a fluorescence lifetime spectrometer. [Fluorescence lifetime spectrometers based on Time Correlated Single Photon Counting (TCSPC) have long suffered from the lack of a compact, broadband excitation source. The source should ideally emit picosecond pulses with a repetition rate adjustable up to megahertz. Supercontinuum sources are an ideal candidate.] Various commercial supercontinuum sources were evaluated. Initial work was carried out on a source with a standard endlessly single-mode photonic crystal fibre (PCF) with a short wavelength limit of 450 nm, followed by later work with a source using high- PCF (a PCF with a large air filling fraction), that had an emission down to 400 nm, and a prototype source with a tapered fibre that had emission to ~320 nm. Key parameters including pulse width, pulse position and pulse height distribution were found to be very wavelength dependent, and their behaviour is explained by theory. The measured pulse widths of the supercontinuum sources were found to be typically ~100 ps, with longer durations found at the blue extreme of the spectra. Analysis of the data showed that this was due to the broadened pulses being a superposition of two pulse sequences with different dispersion characteristics. It was shown that, by taking account of the particular optical and temporal properties of a supercontinuum source, it was possible to make high quality fluorescence lifetime measurements of standard fluorophores such as fluorescein, anthrascene and erythrosine B. A novel device was constructed and evaluated for the wavelength separation of a supercontinuum source based on wedge interference filters. Initial prototypes of the device were able to measure the fluorescence emission spectra of common fluorophores and adequately separate a supercontinuum. Further iterations of the design, employing multiple filters, allowed for the construction of a device that included bandwidth control. The device allowed the transmission bandwidth to be tuned from ~6 nm to >50 nm with a transmission of >70 % for bandwidths >8 nm. The transmission figures achieved are better than any alternative form of wavelength separation, for example devices based on acousto-optic tunable filters (AOTFs) and diffraction gratings. A novel monochromator for fluorescence studies was also constructed using a wedge interference filter simultaneously with a diffraction grating. The design had improved performance compared to a single grating based monochromator in terms of stray light, with only a small drop in throughput and no change in instrument footprint. The stray light performance was found to be comparable to that of a double monochromator over the spectral range of the filter.