Abstract
For quantitative phase imaging (QPI) based on transport-of-intensity equation (TIE), partially coherent illumination provides speckle-free imaging, compatibility with brightfield microscopy, and transverse resolution beyond coherent diffraction limit. Unfortunately, in a conventional microscope with circular illumination aperture, partial coherence tends to diminish the phase contrast, exacerbating the inherent noise-to-resolution tradeoff in TIE imaging, resulting in strong low-frequency artifacts and compromised imaging resolution. Here, we demonstrate how these issues can be effectively addressed by replacing the conventional circular illumination aperture with an annular one. The matched annular illumination not only strongly boosts the phase contrast for low spatial frequencies, but significantly improves the practical imaging resolution to near the incoherent diffraction limit. By incorporating high-numerical aperture (NA) illumination as well as high-NA objective, it is shown, for the first time, that TIE phase imaging can achieve a transverse resolution up to 208 nm, corresponding to an effective NA of 2.66. Time-lapse imaging of in vitro Hela cells revealing cellular morphology and subcellular dynamics during cells mitosis and apoptosis is exemplified. Given its capability for high-resolution QPI as well as the compatibility with widely available brightfield microscopy hardware, the proposed approach is expected to be adopted by the wider biology and medicine community.
Highlights
In the field of optical microscopy, there has been a continued need towards increasing imaging resolution for visualizing subcellular features of the biological samples
Two individual daughter cells are individualized and spread out. These results demonstrate that AI-transport of intensity equation (TIE) is capable of imaging unlabeled cells in the traditional environment of an inverted microscope, allowing for high resolution quantitative phase imaging (QPI) over an extended period of time
The effect of source distribution on the formation of phase information in a partially coherent microscope has been explored, and it is shown that the use of annular illumination matching the objective numerical aperture (NA) allows for high-quality, low-noise phase reconstruction with a lateral resolution close to the microscope incoherent diffraction limit
Summary
I(x) microscope with appropriate annuli fitted into the condenser turret. The 208 nm transverse resolution achieved by combining high-NA annular illumination with high-NA objective detection reveals subcellular structures at high resolution in buccal epithelial cells. Time-lapse imaging of in vitro Hela cell samples is presented, highlighting subcellular dynamics in mitosis and apoptosis in a non-invasive and label-free manner. The experimental results suggest that the developed AI-TIE is a promising, non-destructive, non-interventional tool for structural and functional cellular investigations. Because the approach can be implemented in widely accessible brightfield microscopy hardware, it is expected to be adopted on a large scale by non-specialists, such as bio/pathologists
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