Real-time halo correction in phase contrast imaging

Abstract

As a label-free, nondestructive method, phase contrast is by far the most popular microscopy technique for routine inspection of cell cultures. Yet, features of interest such as extensions near cell bodies are often obscured by a glow, which came to be known as the halo. Advances in modeling image formation have shown that this artifact is due to the limited spatial coherence of the illumination. Yet, the same incoherent illumination is responsible for superior sensitivity to fine details in the phase contrast geometry. Thus, there exists a trade-off between high-detail (incoherent) and low-detail (coherent) imaging systems. In this work, we propose a method to break this dichotomy, by carefully mixing corrected low-frequency and high-frequency data in a way that eliminates the edge effect. Specifically, our technique is able to remove halo artifacts at video rates, requiring no manual interaction or a priori point spread function measurements. To validate our approach, we imaged standard spherical beads, sperm cells, tissue slices, and red blood cells. We demonstrate the real-time operation with a time evolution study of adherent neuron cultures whose neurites are revealed by our halo correction. We show that with our novel technique, we can quantify cell growth in large populations, without the need for thresholds and calibration.

Network science characteristics of brain-derived neuronal cultures deciphered from quantitative phase imaging data

“…Half the media was aspirated twice a week and replaced with fresh maintenance media warmed to 37 °C. Live-cell imaging took place three days in vitro 35 .…”

“…When imaging neural networks, we attempt to ameliorate phototoxicity concerns by reducing the illumination intensity (Thorlabs MCWHL5, 30 milliamps, 3% of total power) and average over several images following the hybrid denoising scheme in 36 . To boost the sensitivity of our measurements, we choose to use Spatial Light Interference Microscopy (SLIM Pro, Phi Optics) which is particularly well suited to imaging the fine details found in neuronal arbors 35 .…”

Reproductive outcomes predicted by phase imaging with computational specificity of spermatozoon ultrastructure

“…The sensitivity in SLIM comes at the expense of a coherence-related halo-artifact present in phase-contrast geometries, which is removed by a Hilbert transform-based technique (Fig. 2B) (38,39).…”

“…For each sperm cell, we traced the sharp discontinuity between the background and the cell, separated by an abrupt change in phase shift. An alternative strategy is to annotate the ultrastructure using immunochemical stains (71) or genetic engineering (39,71,72), at the expense of potential artifacts, more complicated specimen preparation protocol, and difficulty in maintaining genetically modified livestock (73).…”

Wolf phase tomography (WPT) of transparent structures using partially coherent illumination

“…The halo artifacts associated with phase-contrast and SLIM images were removed from the final RI maps using our previously reported algorithm 52 .…”