High light field confinement for fluorescent correlation spectroscopy using a solid immersion lens

Biosens Bioelectron. 2004 Oct 15;20(3):431-5. doi: 10.1016/j.bios.2004.02.028.

Abstract

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.

Publication types

  • Comparative Study
  • Evaluation Study

MeSH terms

  • Equipment Design
  • Equipment Failure Analysis
  • Image Enhancement / instrumentation*
  • Lenses*
  • Microscopy, Confocal / instrumentation*
  • Microscopy, Confocal / methods
  • Microscopy, Fluorescence / instrumentation*
  • Microscopy, Fluorescence / methods
  • Sensitivity and Specificity
  • Spectrometry, Fluorescence / instrumentation*
  • Spectrometry, Fluorescence / methods