Quantitative phase microscopy: automated background leveling techniques and smart temporal phase unwrapping.

In order for time-dynamic quantitative phase microscopy to yield meaningful data to scientists, raw phase measurements must be converted to sequential time series that are consistently phase unwrapped with minimal residual background shape. Beyond the initial phase unwrapping, additional steps must be taken to convert the phase to time-meaningful data sequences. This consists of two major operations both outlined in this paper and shown to operate robustly on biological datasets. An automated background leveling procedure is introduced that consistently removes background shape and minimizes mean background phase value fluctuations. By creating a background phase value that is stable over time, the phase values of features of interest can be examined as a function of time to draw biologically meaningful conclusions. Residual differences between sequential frames of data can be present due to inconsistent phase unwrapping, causing localized regions to have phase values at similar object locations inconsistently changed by large values between frames, not corresponding to physical changes in the sample being observed. This is overcome by introducing a new method, referred to as smart temporal unwrapping that temporally unwraps and filters the phase data such that small motion between frames is accounted for and phase data are unwrapped consistently between frames. The combination of these methods results in the creation of phase data that is stable over time by minimizing errors introduced within the processing of the raw data.