Full-field optical sectioning and three-dimensional localization of fluorescent particles using focal plane modulation.

We present a technique for imaging fluorescent particles based on the axial modulation of the objective's focal plane position. This technique provides full-field optical sectioning and can be used to localize the fluorophores in three dimensions. We describe the technique and apply it to image 200 nm diameter fluorescent beads immobilized in a gel. We show that full-field optical sectioning is obtained and that the beads are localized with a precision of 10 nm in the transverse plane and 14 mn in the axial direction. (c) 2006 Optical Society of America.