Active drift stabilization in three dimensions via image cross-correlation

Abstract

By monitoring stage drift via the normalized cross-correlation of an image of a stuck bead, obtained in real-time, with an out-of-focus "template" image of a similar immobile bead, stored in memory, we implement a simple approach to actively stabilize drift in all three dimensions for existing video microscopy setups. We demonstrate stability to 0.0062 nm along the Z-axis and 0.0031 nm along the X- and Y-axes for long (100 s) timescales.

The tethering of chromatin to the nuclear envelope supports nuclear mechanics

“…To assess nuclear mechanics over a broad range of timescales, we begin by sinusoidally driving the stage with slow oscillations (0.01–0.5 Hz) that recapitulate the timescale of MT polymerization in vivo 36 and then incrementally increase the frequency of the oscillations on a single nucleus to timescales much faster than MT polymerization rates (1–2 Hz). Importantly, we expended great efforts to improve drift control methods that allow us to obtain reliable data at these biologically relevant timescales, which are often inaccessible by force spectroscopy 50 . We first chose to test oscillation amplitudes that drive 50–60 nm nuclear deformations, which approximately recapitulate the scale of MT-dependent NE fluctuations in vivo ( Supplementary Fig.…”

“…To enable high-precision measurements for extended durations, we have implemented a drift correction procedure 50 . Specifically, two template images are acquired, both consisting of a window about an isolated surface-attached bead.…”

A user-friendly two-color super-resolution localization microscope

“…3). Using LabVIEW software, the Normalized crosscorrelation (NCC) graph which represents the likelihood between the current image and the reference image [16,17] is calculated from a cropped region of scatter image that has higher SNR. The motion of sample is detected by tracking the centroid of the NCC graph.…”

“…To evaluate the locking performance we deposited a sparse layer of 1.2μm polystyrene beads fixed on the coverslip, allowing us to measure any movement between the microscope objective and the sample stage. These beads generate strong scattering signal with SNR>10 allowing NCC tracking with high accuracy [17]. The sample movements without and with the active locking are then measured by tracking on one of the beads, as shown in Fig.…”