High-Fidelity Scanning-Free Lensless Microscopy via Adaptive OPD-Domain Fusion for Live-Cell and Tissue Imaging

Multi-wavelength lensless microscopy enables high-speed, wide-field, and high-throughput imaging, making it highly attractive for modern biomedical applications. However, its practical performance is often limited by unreliable autofocusing and wavelength-dependent phase inconsistencies, which together degrade reconstruction fidelity in complex environments. To explicitly address these two limitations, we present a fully scanning-free computational microscopy framework using a static four-wavelength Light-Emitting Diode (LED) illumination module that sequentially switches between wavelengths to provide strong spectral constraints. For robust geometric parameter estimation, we develop an Adaptive-Weighted Multi-wavelength Autofocus (A-WMAF) scheme that exploits the differential defocus sensitivities of multiple wavelengths to yield a single, sharply peaked autofocus curve and thereby reliably determines the sample–sensor distance. To mitigate chromatic phase inconsistencies, we further introduce an iterative optical-path-difference (OPD)–domain adaptive fusion strategy that fuses multi-wavelength phase estimates in a physically consistent OPD space, suppressing wavelength-dependent artifacts and reconstruction noise. With only four raw holograms acquired within seconds, the proposed method achieves high-fidelity quantitative phase reconstruction with a Phase Structural Similarity Index Measure (SSIM) of 0.9942 and a quantitative OPD accuracy of 95.0%, as well as a measured lateral resolution of 1.23 µm, surpassing the Nyquist–Shannon sampling limit. Experimental demonstrations on fixed biological samples and long-term live-cell monitoring validate that the proposed framework simultaneously achieves reliable autofocusing and chromaticity-robust phase fusion, highlighting its potential for high-throughput biomedical imaging and clinical diagnostics.