OBSERVATION OF REAL TIME SYNTHESIS OF INDIVIDUAL DNA MOLECULES

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The equations expressing the intensity of illumination and the overall point-spread function (PSF) of a two-lens system, from Richards and Wolf (1959), were solved numerically, and the effect of a detector aperture was considered. The effects of the numerical aperture, of the radius of the detector aperture, of overfilling or underfilling the objective lens back-aperture, and of using one- or two-photon excitation, on the overall PSF and on the collection volume, were considered. Preliminary results show that the effect of underfilling the objective lens to 80% or higher has little effect on the profile or dimensions of the collection volume. These results are compared with calculations using the paraxial theory and pseudoparaxial theory to describe the light at the focus of the same lens system. These results do not explain the apparently large values of the structure factor (focal volume axial to radial aspect ratio) obtained in fluorescence correlation spectroscopic (FCS) measur ements in our laboratory (Petra Schwille, Sam Hess, unpublished results, 1998). B. Richards and E. Wolf, "Electromagnetic diffraction in optical systems. II. Structure of the image field in an aplanatic system," Proc. R. Soc. London Ser. A 253, 358-379 (1959).