Sample drift estimation method based on speckle patterns formed by backscattered laser light.

ABSTRACT

Single molecule localization microscopy (SMLM) has been established to acquire images with unprecedented resolution down to several nanometers. A typical time scale for image acquisition is several minutes to hours. Yet it is difficult to avoid completely sample drift for long time measurements. To estimate drift, we present a method based on the evaluation of speckle patterns formed by backscattered laser light from the cells using a single molecule localization microscope setup. A z-stack of unique speckle patterns is recorded prior to the measurements as a three-dimensional position reference. During the experiment, images of scattered laser light were acquired, and correlated individually with each of the images of the speckle reference stack to estimate x, y and z drift. Our method shows highly comparable results with a fiducial marker approach, achieving a precision of several nanometers. This method allows for high precision three dimensional drift correction of microscope systems without any additional sample preparation.