| Fluorescence microscopy is one of the most widely used and versatile techniques in cell and molecular biology. As an imaging technique its benefits include the non-invasive nature of light which allows the observation of living samples, and specific fluorescent labeling with genetically encoded or exogenous probes enabling a particular molecular component of the sample to be visualized. The spatial resolution of light microscopy is a significant limitation, however. Sub-cellular structures and molecular complexes essential for biological function exist on length scales from nanometers to micrometers. When observed with light, structural features smaller than ∼0.2 mum are blurred and difficult or impossible to resolve. In this thesis we introduce a new concept for fluorescence microscopy which circumvents the classical resolution limit. Our approach is generally applicable to biological imaging and requires relatively simple experimental apparatus. We demonstrate a ten-fold resolution improvement over conventional optical microscopy.;In Chapter 2 of the thesis we describe the discovery and characterization of a novel fluorescence switching effect. We found that single molecules of a red carbocyanine dye can be repeatedly switched between a fluorescent state and a "dark", non-fluorescent state over many cycles, under the control of an external light source. We quantitatively characterized the effect and present preliminary data investigating its photo-physical and chemical mechanism.;Stochastic Optical Reconstruction Microscopy (STORM), a new method for fluorescence microscopy with sub-diffraction-limit spatial resolution, is introduced in Chapter 3. The principle of this method is the precise determination of the positions of individual fluorescent molecules labeling the sample. The fluorescent signal from each label is isolated and measured. This isolation is achieved by taking advantage of photo-switchable fluorophores whose fluorescence emission can be switched on and off in time. Using this method we demonstrate an image resolution of ∼20 nm for biomolecular structures in vitro and in cell cultures.;In Chapter 4 we discuss the extension of STORM to multi-color imaging and sub-diffraction-limit resolution in three dimensions. New photo-switchable fluorescent probes with distinct spectral properties are introduced which enable the simultaneous visualization of multiple cellular targets. Also, a method for measuring the axial positions of individual fluorophores is combined with our imaging technique to generate 3D super-resolution images of fluorescently labeled biological samples. |
