High light field confinement for Fluorescence Correlation Spectroscopy using a Solid Immersion Lens

In this paper we present recent single molecule detection experiment using a solid immersion lens (SIL) for fluorescent correlation spectroscopy measurements. We compared the performance of the SIL in combination with an air objective (40×, numerical aperture (NA)=0.6) with a water immersion objective (40×, NA=1.15) in a confocal microscope system (ConfoCorr 1). Important parameters for single molecule experiments such as collection efficiency and excitation field confinement were investigated. Although the two set-ups have similar numerical aperture the measurements demonstrated higher field confinement and better collection efficiency for the SIL system in comparison to the conventional confocal set-up. Adding spherical aberrations shifts the sample volume up to 4 μm away from the plane surface of the SIL and conserves a diffraction limited focal volume. In this case the FCS autocorrelation demonstrates a free 3D diffusion of dye molecules in a highly confined light field.