|dc.description.abstract||Total internal reflection fluorescence microscopy (TIRFM) techniques play an important role in investigating phenomena which occur on transparent dielectric surfaces at nanometre scales in various research fields from molecular biology to medical devices. In many cases TIRFM is used to estimate or measure distances where a precise knowledge of the penetration depth (dp) is needed. There is usually a misleading assumption that the detected fluorescence is a pure product of the uniform near-field excitation. Therefore, a critical issue in TIRFM is to get accurate information about the precise dp or evanescence wave (EW) profile which in turn depends on the angle of incidence (AOI). I have developed a novel 3D-printable alignment jig (A-JIG) for the facile and precise measurements of the AOI at a variety of excitation wavelengths for through-the-objective TIRFM systems. This device is capable of measuring AOIs of various excitation wavelengths at the same time, above and below the critical angle which is especially important for basic, multi-colour, variable-angle (VA), and multi-angle (MA) TIRFM applications. The A-JIG is compatible with coverslips of different thicknesses and refractive indices. Moreover this device also works as a sampling stage, therefore providing the ability to measure or alter AOIs at any stage of the experiment. The importance of this study lies in the fact that AOI is one of the main factors influencing dp of the EW.
Since EW is an important feature responsible for fluorescence excitation in all TIRFM systems there is a continuous need for the development of novel, better, universal, rapid and precise standards for its calibration in a reproducible manner. Therefore I developed a solid state standard (Chapter 4), as a versatile, simple and almost costless alternative to the commercially available standards for in situ EW calibration using a metal sphere. The dp of the EW as a function of diameter of an imaged metal sphere was evaluated. The analysis of inverted images of the metal sphere immersed in aqueous fluorescein isothiocyanate isomer I (FITC) was performed entirely with ImageJ free software. Comparison of the dp measurements obtained using the presented standard was in agreement with theoretically predicted values.
In Chapter 5 and 6 a rapid and accurate TIRFM based methodology for the in situ physiochemical characterization of monolayers and their adsorption processes on modified borosilicate glass surfaces is presented. The measurements were based on the use of a TIRFM system modified with a portable spectrometer (Ocean Optics USB4000-FL). This apparatus was utilized for studying both, adsorption isotherms, and spectral properties of monolayers of FITC labelled bovine serum albumin (FITC-BSA) and FITC at pH 5.0, 7.4, and 9.6. The glass surfaces were modified and chemically functionalized to alter their wettability properties. Both types of molecules easily adsorbed on hydrophobic and unmodified flat surfaces forming a stable and uniform coating in less than 30 minutes. Emission intensity, spectra, and photobleaching profiles of FITC and BSA-FITC monolayers were all dependent on both the solution pH and the hydrophobic/hydrophilic properties of the glass surfaces. At pH 7.4 and 9.6 on hydrophobic surfaces FITC monolayers were bright (due to dominant dianion form of the FITC) and reproducibly formed, with well defined, fast photobleaching kinetics (decaying up to ~50% intensity in the first 5 minutes of imaging). However the BSA-FITC monolayers were brighter, more photostable, and had different photobleaching profiles which was related to the different environment when attached to the BSA. No or residual adsorption was detected for hydrophilic surfaces.
Unexpectedly, at pH 5.0 on hydrophobic surfaces, FITC monolayers were both bright and apparently un-bleachable over 20 minutes of imaging. I assume that during the adsorption process, which was driven by a hydrophobic-hydrophobic interaction, there was conversion of the neutral FITC form to its fluorescent quinoid isomer. During monolayer formation at this pH I saw clear evidence for both concentration-based quenching which indicated high surface coverage, and unusual emission recovery indicative of some form of species equilibrium on the surface. While the mechanism of this strange behaviour is still unknown, the data shows that the monolayers were highly reproducible and exhibit similar spectral properties to their analogues in the aqueous solution, making them potentially useful in various applications. All monolayers were easily prepared, low cost, and can serve as convenient test samples for TIRFM alignment, calibration, and validation prior to undertaking measurements with more sensitive or expensive biological specimens. The high repeatability between measurements indicates that this methodology is facile and rapid for measuring properties of the fluorescence emitting monolayers which is important in many areas of medical devices, biological sensors and biomaterials research.||en_IE