Fluorescence correlation spectroscopy at 100 nM concentrations using near-field scanning optical microscopic (NSOM) geometries and highly diffracting force sensing fiber probes

A tapered cantilevered optical fiber probe is introduced for fluorescence correlation spectroscopy (FCS). The probe has an aperture that is coated with metal and its dimension can be very well defined. Its design is based on cantilevered near-field optical fiber probes that allow for on-line atomic force feedback. This permits unprecedented stability in FCS measurements. Even such probes that have large diameters are below the cutoff wavelength of light propagation in optical fibers. Such large diameter probes produce bright spots of light that are highly diffracting. Therefore, only the fluence of light very close to the surface of the glass probe aperture is sufficient to exhibit a high probability of fluorescence excitation. From the results presented in this paper the z extent of such probes seems comparable to what can be obtained from prism-based evanescent wave methods, but with much more flexibility and including force sensing capability. In agreement with theoretical results on prism evanescent field-based excitation, it is shown that even with large xy dimensions for the fiber probes in this paper, fluorescence correlation spectra from 100 nM fluorophore concentrations can be recorded. This is 10 times larger than the concentrations generally required for conventional confocal FCS. The results suggest that such an approach has considerable potential for applications of FCS in membrane, near-membrane, solution, and even within-cell environments. The latter is achieved through atomic force feedback controlled penetration of tapered cantilevered metal-coated glass fibers into cells, and this is also reported in this paper.