As a label-free imaging technique, quantitative phase imaging (QPI) precisely measures the phase delay induced by a sample, thereby enabling the extraction of key parameters such as geometric thickness and refractive index. It holds significant value in materials characterization, cell biology, and medical diagnostics. In recent years, surface plasmon resonance holographic microscopy (SPRHM) has combined the near-field optical phenomenon of surface plasmon resonance (SPR) with digital holographic microscopy, achieving non-invasive, high-sensitivity near-field QPI. This technique has been widely applied in the characterization of two-dimensional materials and dynamic monitoring of cell-substrate adhesion.
However, in near-field QPI, factors such as optical component manufacturing defects, system misalignments, off-axis interference between object and reference beams, and particularly environmental disturbances introduce complex, time-varying wavefront aberrations. These aberrations pose a critical bottleneck, severely limiting the in-depth application of near-field QPI and hindering high-sensitivity quantitative characterization of sample parameters.
Existing aberration correction methods fall into two main categories: physical compensation in the optical path and numerical post-processing. The former requires precise adjustments or additional components, while the latter often involves computationally intensive procedures or lacks flexibility. Although the double-exposure (DE) method can eliminate background phase aberrations, it relies on recording images without a sample, making it unsuitable for scenarios with high sample density, immobile samples, or continuously fluctuating aberrations. Therefore, developing rapid, accurate, and in-situ wavefront aberration correction methods is crucial for advancing near-field QPI and its applications.
In a new paper in Light: Science & Applications , Prof. Jianlin Zhao, Dr. Siqing Dai, and their team at Northwestern Polytechnical University, in collaboration with Prof. Gerd Ulrich Nienhaus of Karlsruhe Institute of Technology, propose adaptive optics (AO)-based SPRHM for effective removal of spatiotemporal phase aberrations and high-quality quantitative phase imaging. This method requires no wavefront sensor; instead, it leverages the inherent advantage of digital holography—the ability to measure complex amplitude—to enable real-time measurement and compensation of near-field wavefront aberrations.
The method integrates digital holographic microscopy (DHM) with a spatial light modulator (SLM) to perform in-situ, fast, and flexible aberration correction. By decomposing wavefront distortions into low-order Zernike modes and optimizing their coefficients using heuristic algorithms (e.g., hill climbing, particle swarm optimization), the system pre-compensates phase aberrations before they distort the sample image. This approach eliminates the need for separate reference measurements or wavefront sensors, relying instead on background regions within the same field of view.
Experimentally, the authors validated AO-SPRHM using an electron-beam lithography (EBL)-structured photoresist test sample and live human breast cancer cells (MDA-MB-231). In both cases, raw SPR phase images showed severe background distortions, which were effectively removed by all five implemented optimization algorithms. The corrected images exhibited flat backgrounds and high contrast, with Zernike coefficients dominated by tip and tilt modes. For live cells, AO-SPRHM produced adhesion gap maps comparable to the DE method but without the need for sample removal or sequential acquisition. Long-term imaging of osteoblast cells over 10 hours demonstrated that AO-SPRHM maintains stable background correction, while DE gradually degraded due to time-varying aberrations. The method enabled quantitative analysis of cell detachment dynamics, including projected area, gap volume, and surface area over time.
The discussion highlights that AO-SPRHM is robust even with dense cell layers, provided at least ~35% background area is available. Higher-order Zernike modes beyond the first six introduced artifacts, indicating that mode selection must be tailored to the optical system. The hill climbing algorithm offered the best trade-off between speed and accuracy. Limitations include the need for background regions and potential overfitting, though regularization and stopping rules mitigate this.
In terms of future applications, the authors propose extending AO-SPRHM to transmission DHM, total internal reflection holography, and incoherent computational adaptive optics. Dual-wavelength operation could enable simultaneous measurement of cell-substrate distance and refractive index. The method is particularly promising for long-term, label-free monitoring of cellular dynamics, nanoparticle analysis, and 2D material characterization, offering a generalizable platform for adaptive QPI in biomedical and materials science.
Light: Science & Applications
Background-free quantitative phase imaging with adaptive-optics surface plasmon resonance holographic microscopy