Fluorescence microscopy and spectroscopy have been successfully utilized in a range of biological applications. However, challenges still remain due to endogenous background signal that obscures measurements and spatial resolution that is limited by excitation and emission wavelengths. This work describes sensitivity and resolution improvements to fluorescence-based measurements through a combination of optical modulation and correlative methods including fluorescence correlation spectroscopy (FCS), synchronously amplified fluorescence image recovery (SAFIRe) and super-resolution optical fluctuation imaging (SOFI). First, optical modulation is combined with FCS, utilizing subtraction of correlation functions to obtain a background subtracted FCS signal. Next, background reduction is accomplished through pulsed excitation and is subsequently is applied to FCS to demonstrate background reduction. Resolution improvements beyond the Abbe limit of diffraction are also explored, employing the stochastic blinking of fluorophores in a point scanning geometry, and optimal fluorophore photophysical characteristics are identified. Finally, an instrument for tracking single fluorescent particles in real time is described which could provide new insights into freely diffusing rapid intramolecular interactions with eventual application in biological systems.
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Improved sensitivity in fluorescence spectroscopy and microscopy
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