Stabilizing method for reflection interference contrast microscopy.

Reflection interference contrast microscopy (RICM) is a technique for measuring the shape and position of microscopic objects in solution; it has many biological and biophysical applications. Use of RICM for long-time acquisitions requires minimizing defocusing effects that are due to thermal and mechanical drift. We present a simple stabilizing method that accomplishes this using an image-analysis-based linear focus function to establish feedback control of the focal position. While implementing this routine, we used RICM for independent measurement of the apparent fluctuation in the vertical position of an immobilized bead: the measured height had a standard deviation of 0.12 nm during a 45 min acquisition while under feedback control, demonstrating the high stability achievable with our approach.