A novel method of focus sensing for the laser scanner microscope.

An unconventional approach to the focus-sensing problem in a high-speed cell scanner has resulted in a method of directly detecting the position of the cell layer. This is done by using two coaxial but axially shifted beams, one focused on either side of the cell layer. The modulation induced in each beam by the cell's fine structure is compared electronically to yield the focus-error signal. The beams are orthogonally polarized, therefore separable, and are independently detected. The two detector-output signals are used to form both the focus-error signal and the scanner's primary data signal. This focus-sensing technique has been analytically modeled and experimentally demonstrated for a high-numerical-aperture system. In that system, the depth of focus is less than the thickness of the cell layer. It is shown that a lens fabricated from a birefringent crystal is an attractive means of forming the required orthogonally polarized axially shifted beams.