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Fan Y, Zheng CY, Shu YF et al. Aberration-corrected differential phase contrast microscopy with annular illuminations. Opto-Electron Sci x, 240037 (2025). doi: 10.29026/oes.2025.240037
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Article Open Access
Aberration-corrected differential phase contrast microscopy with annular illuminations
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Abstract
Quantitative phase imaging (QPI) enables non-invasive cellular analysis by utilizing cell thickness and refractive index as intrinsic probes, revolutionizing label-free microscopy in cellular research. Differential phase contrast (DPC), a non-interferometric QPI technique, requires only four intensity images under asymmetric illumination to recover the phase of a sample, offering the advantages of being label-free, non-coherent and highly robust. Its phase reconstruction result relies on precise modeling of the phase transfer function (PTF). However, in real optical systems, the PTF will deviate from its theoretical ideal due to the unknown wavefront aberrations, which will lead to significant artifacts and distortions in the reconstructed phase. We propose an aberration-corrected DPC (ACDPC) method that utilizes three intensity images under annular illumination to jointly retrieve the aberration and the phase, achieving high-quality QPI with minimal raw data. By employing three annular illuminations precisely matched to the numerical aperture of the objective lens, the object information is transmitted into the acquired intensity with a high signal-to-noise ratio. Phase retrieval is achieved by an iterative deconvolution algorithm that uses simulated annealing to estimate the aberration and further employs regularized deconvolution to reconstruct the phase, ultimately obtaining a refined complex pupil function and an aberration-corrected quantitative phase. We demonstrate that ACDPC is robust to multi-order aberrations without any priori knowledge, and can effectively retrieve and correct system aberrations to obtain high-quality quantitative phase. Experimental results show that ACDPC can clearly reproduce subcellular structures such as vesicles and lipid droplets with higher resolution than conventional DPC, which opens up new possibilities for more accurate subcellular structure analysis in cell biology. -
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References
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Supplementary information for Aberration-corrected differential phase contrast microscopy with annular illuminations 
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Fan Y, Zheng CY, Shu YF et al. Aberration-corrected differential phase contrast microscopy with annular illuminations. Opto-Electron Sci x, 240037 (2025). doi: 10.29026/oes.2025.240037
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Figure 1.
PTF with ideal pupil and complex pupil under different illuminations. (a) Ideal pupil without aberration and complex pupil with aberration for simulation. (b) Comparison of PTFs with ideal pupil and complex pupil under single point illumination at different angles. (c) Comparison of PTFs with ideal pupil and complex pupil under half-circular and half-annular (NAill = NAobj) illuminations.
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Figure 2.
The flowchart of ACDPC iterative reconstruction joint optimization algorithm. (a) ACDPC imaging system based on half-annular illumination. (b) Three annular illumination patterns and their intensity images. (c) The flowchart of ACDPC method.
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Figure 3.
Simulation of DPC and ACDPC reconstructed phases with different aberrations. (a) Reconstructed phases of DPC and ACDPC with different degrees of defocus aberration. (b) Original and retrieved pupils and corresponding reconstructed phase details in defocus aberration. (c) Average number of convergence and phase RMSE data for ten aberrations. (d) Reconstructed phases of DPC and ACDPC with multi-order aberration. (e) Ground-truth pupil and corresponding PTF and ACDPC retrieved pupil and PTF. (f) Quantitative profile comparison of reconstructed phase. (g) Zernike coefficients for ground-truth and retrieved aberrations.
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Figure 4.
Experimental results of USAF resolution targets. (a) ACDPC experimental system based on commercial inverted microscope with annular illumination. (b) The actual acquired intensity image and the zoom-in image of region of interest (ROI 1). (c) Full-field-of-view reconstructed phase and zoom-in images (ROI 1) of DPC and ACDPC at −10 μm defocus, and quantitative phase profile. (d) Reconstructed phase and zoom-in images (ROI 2) of DPC and ACDPC at +10 μm defocus. (e) Reconstructed phase of DPC and ACDPC at +20 μm defocus and the retrieved aberration coefficient.
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Figure 5.
Experimental results of DPC and ACDPC on NIH 3T3 cells. (a) Experimental result of DPC and ACDPC experiments on NIH 3T3 cell samples under fine focusing conditions. (b) Magnified images of two ROIs (ROI 1, ROI 2) in (a). (c) Zernike coefficient of retrieved aberration in (b). (d) Experimental result of DPC and ACDPC experiments on NIH 3T3 cell samples under coarse focusing conditions. (e) Magnified image of the ROI (ROI 1) in (a) and further magnification of its ROI (ROI 2). (f) Zernike coefficient of retrieved aberration in (e).
