Quantitative phase imaging verification in large field-of-view lensless holographic microscopy via two-photon 3D printing

Abstract: Large field-of-view (FOV) microscopic imaging with high lateral resolution (1-2 microns for high space-bandwidth product) plays a pivotal role in biomedicine and biophotonics, especially within the label-free regime, e.g., for whole slide tissue quantitative analysis and live cell culture imaging. In this context, lensless digital holographic microscopy (LDHM) holds substantial promise. However, one intriguing challenge has been the fidelity of computational quantitative phase imaging (QPI) with LDHM in large FOV. While photonic phantoms, 3D printed by two-photon polymerization (TPP), have facilitated calibration and verification in small FOV lens-based QPI systems, an equivalent evaluation for lensless techniques remains elusive, compounded by issues such as twin-image and beam distortions, particularly towards the detector edges. To tackle this problem, we propose an application of TPP over large area to examine phase consistency in LDHM. In our research, we crafted widefield calibration phase test targets, fabricated them with galvo and piezo scanning, and scrutinized them under single-shot twin-image corrupted conditions and multi-frame iterative twin-image minimization scenarios. By displacing the structures toward the edges of the sensing area, we verified LDHM phase imaging errors across the entire field-of-view, showing less than 12 percent of phase value difference between investigated areas. Interestingly, our research revealed that the TPP technique, following LDHM and Linnik interferometry cross-verification, requires novel design considerations for successful large-area precise photonic manufacturing. Our work thus unveils important avenues toward the quantitative benchmarking of large FOV lensless phase imaging, advancing our mechanistic understanding of LDHM techniques and contributing to their further development and optimization of both phase imaging and fabrication.