High light field confinement for fluorescent 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 (40x, numerical aperture (NA)=1.15) with a water immersion objective (40x, NA=0.6) 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 microm 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.
Serov
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A
Institute for Biomedical Imaging, Optics, and Engineering of the Swiss Federal Institute of Technology (EPFL), CH-1015 Lausanne, Switzerland. alexandre.serov@epfl.ch
Rao
R
R
Gösch
M
M
Anhut
T
T
Martin
D
D
Brunner
R
R
Rigler
R
R
Lasser
T
T
D004867:Equipment Design; D019544:Equipment Failure Analysis; D007089:Image Enhancement / Q000295:instrumentation*; D007909:Lenses; D018613:Microscopy, Confocal / Q000295:instrumentation* / Q000379:methods; D008856:Microscopy, Fluorescence / Q000295:instrumentation* / Q000379:methods; D012680:Sensitivity and Specificity; D013050:Spectrometry, Fluorescence / Q000295:instrumentation* / Q000379:methods