Academic literature on the topic 'Fluorescence imaging systems'

This thesis describes the application of multidimensional fluorescence imaging to microfluidic systems. The work focuses on time- and polarisation-resolved fluorescence microscopy to extract information from microchannel environments. The methods are applied to polymerase chain reaction (PCR) and a DNA repair enzyme, uracil DNA glycosylase (UDG). The fluorescence lifetimes Rhodamine B are calibrated over a thermal gradient using time correlated single photon counting. The dye is then introduced in solution into a novel microfluidic PCR device. Fluorescence lifetime imaging microscopy (FLIM) is then performed, and using the calibration curve, the temperature distributions are accurately determined. The device is subsequently optimised for efficient DNA amplification. A line-scanning FLIM microscope is used to characterise a rapid microfluidic mixer via a fluorescence quenching experiment. Fluorescein and sodium iodide are mixed in a continuous flow format and imaged in 3-D. The spatial distributions of the fluorescence lifetimes are converted to the concentrations of sodium iodide to quantify mixing. Computational fluid dynamic (CFD) simulations are validated by comparison to the quantitative concentrations obtained experimentally. The binding reaction between UDG and a hexachlorofluorescein (HEX) labelled DNA strand is characterised spectrally. As well as an increase in fluorescence polarisation anisotropy, a 700 ps increase in the fluorescence lifetime is measured. Confocal microscopy shows the same spectral properties when the reaction is performed in both simple and rapid microfluidic mixers. In the latter experiment, a concentration series allows the determination of kinetics, which agree with conventional stopped-flow data. A two-colour two-photon (2c2p) FLIM microscope is developed and applied to the UDG-DNA system. An oligonucleotide containing 2-aminopurine, a reporter of DNA base flipping, and HEX is mixed with UDG in a microfluidic Y-mixer. The 2c2p excitation allows FLIM of both fluorophores and hence detection of binding and base flipping. Comparison to CFD with known kinetic rate constants confirms the experimental observations.

Heptamethine cyanine dyes are attractive compounds for imaging purposes in biomedical applications because of their chemical and photophysical properties exhibited in the near-infrared region. A series of meso amino-substituted heptamethine cyanine dyes with indolenine, benz[e]indolenine and benz[c,d]indolenine heterocyclic moieties were synthesized and their spectral properties including fluorescence quntum yield were investigated in ethanol and ethanol/water mixture. Upon substitution with amines, the absorption maxima of the dyes shifted to the lower wavelength region (~600 nm), showed larger Stokes shifts and stronger fluorescence which can be attributed to an excited state intramolecular charge transfer (ICT). High quantum yields were observed for primary amine derivatives and lower quantum yields were observed for secondary amine derivatives. Fluorescence quantum yields are greater for dyes with 3H-indolenine terminal moieties than for dyes with benz[e]indolenine end groups. Benz[c,d]indolenine based heptamethine cyanine dyes exhibited the lowest quantum yield due to aggregation in solution. In general, the benz[e]indolenine hepatemethine cyanines showed high Stokes shifts compared to indolenine dyes. For the meso-chloro dyes, the absorption maxima for the dyes shifted bathochromically in the order of indolenine, benz[e]indolenine and benz[c,d]indolenine.

Thesis (M.S.E.C.E.)--University of Delaware, 2007.<br>Principal faculty advisors: Fouad Kiamilev and Robert F. Rogers, Dept. of Electrical and Computer Engineering. Includes bibliographical references.

Existing optic fibre-bundle based imaging probes have been successfully used to image biological signals from tissue in direct contact with the probe tip (Hirano et al. 1996). These fibre-bundle probe systems employed conventional fluorescence microscopy and thus lacked spatial filtering or a scanned light source, two features used by laser scanning confocal microscopes (LSCMs) to improve signal quality. Improving the methods of imaging tissue in its natural state, deep in-vivo and at cellular resolution is an ever-present goal in biological research. Within this study, a novel (580 μm diameter) optic fibre-bundle direct-contact imaging probe, employing a LSCM, was developed to allow for improved imaging of deep biological tissue in-vivo. The new LSCM/probe system possessed a spatial resolution of 10 μm, and a temporal resolution of 1 msec. The LSCM/probe system was compared to a previously used direct-contact probe system that employed a conventional fluorescence microscope. Quantitative and qualitative data indicated that the LSCM/probe system possessed superior image contrast and quality. Furthermore, the LSCM/probe system was approximately 16 times more effective at filtering unwanted contaminating light from regions below the imaging plane (z-axis). The unique LSCM/probe system was applied to an exploratory investigation of calcium activity of both glial and neuronal cells within the whisker portion of the rat primary somatosensory cortex in-vivo. Fluorescence signals of 106 cells were recorded from 12 female Sprague Dawley rats aged between 7-8 weeks. Fluo-3(AM) fluorophore based calcium fluctuations that coincided with 10 - 14 Hz sinusoidal stimulation of rat whiskers for 0.5-1 second were observed in 8.5% of cells (9 of 106). Both increases and decreases in calcium levels that coincided with whisker stimulation were observed. Of the 8.5 % of cells, 2.8% (3 cells) were categorized as glial and 5.7% (6 cells) as neuronal, based on temporal characteristics of the observed activity. The remaining cells (97 of 106) displayed sufficient calcium-based intensity but no fluctuations that coincided with an applied stimulus. This was partially attributed to electronic noise inherent in the prototype system obscuring potential very weak cell signals. The results indicate that the novel LSCM/probe system is an advancement over previously used systems that employed direct-contact imaging probes. The miniature nature of the probe allows for insertion into soft tissue, like a hypodermic needle, and provides access to a range of depths with minimal invasiveness. Furthermore, when combined with selected dyes, the system allows for imaging of numerous forms of activity at cellular resolution.

Thesis (Ph. D.)--University of Oregon, 2006.<br>Typescript. Includes vita and abstract. Includes bibliographical references (leaves 103-108). Also available for download via the World Wide Web; free to University of Oregon users.

Fluorescence microscopy allows for spatial and temporal resolution of systems which are inherently fluorescent or which can be selectively labeled with fluorescent molecules. Temporal resolution is crucial for imaging real time processes in living samples. A common problem in fluorescence microscopy of biological samples is autofluorescence, fluorescence inherent to the system, which interferes with detection of fluorescence of interest by decreasing the signal to noise ratio. Two current methods for improved imaging against autofluorescence are two-photon excitation and total internal reflection microscopy. Two-photon excitation occurs when two longer wavelength photons are absorbed quasi-simultaneously by a single fluorophore. For this to take place there must be a photon density on the order of 1030 photons/(cm2)(s), which is achieved through use of a femtosecond pulsed laser and a high magnification microscope objective. Two-photon excitation then only occurs at the focal spot, significantly reducing the focal volume and therefore background autofluorescence. The second method, total internal reflection, is based on evanescent wave excitation, which decreases exponentially in intensity away from the imaging surface. This allows for excitation of a thin (~200 nm) slice of a sample. Since only a narrow region of interest is excited, an optical slice can be imaged, decreasing excitation of out-of-focus autofluorescence, and increasing the signal to noise ratio. By coupling total internal reflection with two-photon excitation, an entire cell can be imaged while still maintaining the use of lower energy photons to irradiate the sample and achieve two-photon excitation along the length traveled by the evanescent wave. This system allows for more sensitive detection of fluorescence of interest from biological systems as a result of a significant decrease in excitation volume and therefore a decrease in autofluorescence signal. In the two-photon total internal reflection microscopy setup detailed in this work, an excitation area of 20 μm by 30 μm is achieved, and used to image FITC-stained actin filaments in BS-C-1 cells

The technique of Fluorescence Lifetime Imaging Microscopy (FLIM) has been employed to quantitatively and spatially map the fluid composition and temperature within microfluidic systems. A molecular probe with a solvent-sensitive fluorescence lifetime has been exploited to investigate and map the diffusional mixing of fluid streams under laminar flow conditions within a microfluidic device. Using FLIM, the fluid composition is mapped with high quantification and spatial resolution to assess the extent of mixing. This technique was extended to quantitatively evaluate the mixing efficiency of a range of commercial microfluidic mixers employing various mixing strategies, including the use of obstacles fabricated within the channels. A fluorescently labelled polymer has been investigated as a new probe for mapping temperature within microfluidic devices using FLIM. Time Correlated Single Photon Counting (TCSPC) measurements showed that the average fluorescence lifetime displayed by an aqueous solution of the polymer depended strongly on temperature, increasing from 3 ns to 13.5 ns between 23 and 38 oC. This effect was exploited using FLIM to provide high spatial resolution temperature mapping with sub-degree temperature resolution within microfluidic devices. A temperature-sensitive, water-soluble derivative of the rhodamine B fluorophore, effective over a wide dynamic temperature range (25 to 91 oC) has been used to map the temperature distribution during the mixing of fluid streams of different temperatures within a microchannel. In addition, this probe was employed to quantify the fluid temperature in a prototype microfluidic system for DNA amplification. FLIM has been demonstrated to provide a superior approach to the imaging within microfluidic systems over other commonly used techniques, such as fluorescence intensity and colourimetric imaging.