Technique development in super-resolution fluorescence microscopy

I present three projects all of which involve innovations in fluorescence microscopy. First I present a microscopy method whereby the angular dependence of a fluorophore's emission pattern near a bare glass surface or metal-coated surface that supports surface plasmon resonance is measured. This technique involves altering the microscope optics to directly record (on a CCD camera) the intensity pattern at the objective's back focal plane. This intensity pattern directly maps the angular emission pattern of fluorescence. The experimental emission profile on both glass and aluminum-coated surfaces is anisotropic with a peak at either the critical angle or both the critical angle and the surface plasmon angle. The observed profiles on both glass and aluminum-coated surfaces are anisotropic and agree well with computer calculations.; Second I present a new fluorescence resonance energy transfer (FRET) method based on polarization that determines FRET using data from a single camera exposure, offering better time resolution of dynamic associations. Polarized FRET uses a simultaneous combination of excitation wavelengths from two orthogonally polarized sources, along with an emission channel tri-image splitter outfitted with appropriate polarizers, to concurrently excite and collect fluorescence from free donors, free acceptors, and FRET pairs. The pixel-by-pixel concentrations of all molecules can then be determined. Here I present the theory of polarized FRET and examine its feasibility through both theoretical investigation and experimental confirmation on mixtures of fluorescent proteins expressed in living cells.; Third, I present a method for directly measuring the depth and purity of the evanescent field used for fluorophore excitation in total internal reflection fluorescence (TIRF) microscopy. This technique involves microscopic observation of low refractive index, fluorescently labeled, spherical beads in an index-matched solution. With both the 1.45 NA and 1.65 NA objectives on the Olympus microscope the profile of the evanescent field fits well to a double exponential with 90% of the field represented by an exponential with a decay rate close to that expected for a pure evanescent field and 10% of the field represented by an exponential with a much longer decay constant attributed to scattering.