In the field of digital pathology and biomedical research, the demand for high-resolution, wide field-of-view (FOV) imaging is paramount. Traditional methods, like whole slide imaging (WSI) scanners, often involve complex hardware, mechanical scanning, and a fundamental trade-off between resolution and FOV. Fourier Ptychographic Microscopy (FPM) emerged as a promising computational alternative, synthesizing a large synthetic aperture from multiple angled illuminations to break the diffraction limit. However, its requirement for hundreds of raw images has been a major bottleneck for efficiency.

To address this critical challenge, KFBIO collaborated with the Smart Computational Imaging (SCI) Laboratory at Nanjing University of Science and Technology to develop a groundbreaking solution: the Efficient Synthetic Aperture for FPM (ESA-FPM). This innovative technology dramatically enhances imaging throughput, making rapid, high-quality digital pathology a practical reality.

The Challenge: Efficiency in High-Throughput Imaging

Conventional FPM achieves high spatial bandwidth by sequentially capturing dozens or even hundreds of low-resolution images under varied coherent illuminations. This process ensures data redundancy for stable algorithmic convergence but results in prolonged acquisition times (often minutes) and large datasets. For time-sensitive applications like intraoperative pathology or high-volume screening, this inefficiency is a significant barrier.

The ESA-FPM Innovation: Hybrid Illumination for Maximum Efficiency

The core breakthrough of ESA-FPM lies in its novel hybrid coherent and incoherent illumination scheme, which maximizes data utility from every captured image.

1. Single-Shot Incoherent Bright-Field Image: Instead of sequential coherent LEDs, ESA-FPM illuminates the sample with all bright-field LEDs simultaneously. This single incoherent image captures sample information across a bandwidth twice that of the coherent diffraction limit of the objective lens in one acquisition.

2. Efficient Centrosymmetric Dark-Field Acquisition: For higher-resolution details, ESA-FPM employs a sparse, centrosymmetric LED illumination pattern. Pairs of opposite LEDs are lit simultaneously. A key theoretical insight is that for stained pathology samples (modeled as weak-phase objects), this symmetric illumination cancels out phase contributions, leaving only the crucial absorption information. Each dark-field image thus updates two symmetric regions in the Fourier spectrum, doubling efficiency.

Through rigorous data redundancy analysis, the team optimized this scheme. The result: ESA-FPM achieves a synthetic aperture of 3x the objective’s NA using only 7 raw images—one bright-field and six dark-field acquisitions.

Unmatched Performance: Experimental Validation

The advantages of ESA-FPM are not just theoretical but proven experimentally:

• Resolution Match with 98.4% Less Data: Using a standard USAF target, ESA-FPM achieved a full-pitch resolution of 776 nm, equivalent to conventional FPM. However, it required only 7 images compared to FPM’s 441, representing a 98.4% reduction in data acquisition.

• Superior Speed: The total acquisition time was slashed from approximately 3 minutes (conventional FPM) to about 1.5 seconds.

• High-Throughput Pathological Imaging: A customized, miniaturized ESA-FPM system was built for pathology, featuring a specialized 6x/0.35 NA objective and a high-brightness LED array. Imaging an H&E-stained lymph node metastasis slide, ESA-FPM produced a high-resolution, high-contrast image across a large FOV of 2.19 x 1.46 mm². It revealed cellular and sub-cellular details (like lymphocyte nuclei) with clarity matching or surpassing a 40x/0.65 NA objective lens, but over an area 44.5 times larger.

Why ESA-FPM is a Game-Changer for Digital Pathology

The collaboration between KFBIO and SCI Lab has yielded a technology that directly addresses the pressing needs of modern pathology:

• Speed: Near-real-time imaging enables rapid diagnosis and screening.

• Simplicity & Cost: Reduces system complexity by minimizing mechanical and control requirements for extensive LED sequencing.

• High Throughput: Delivers both large FOV and high resolution without scanning, perfect for creating comprehensive “digital slides.”

• Robustness: The hybrid model and efficient algorithm ensure stable convergence and high-quality reconstruction from minimal data.

Conclusion: Pioneering the Future of Diagnostic Imaging

The development of ESA-FPM marks a significant leap forward in computational imaging. By rethinking the fundamental illumination and acquisition strategy of FPM, KFBIO and the SCI Lab have created a practical, efficient, and powerful tool tailored for biomedical and pathological applications.

This technology paves the way for next-generation, high-throughput microscopes and scanners that are faster, more compact, and accessible, ultimately accelerating advancements in disease diagnosis, cell biology, and medical research.

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From Lab to Product: Powering KFBIO’s High-Magnification Oil-Immersion-Free Scanner

The groundbreaking ESA-FPM technology is not confined to the research lab; it is the core engine behind KFBIO’s innovative high-magnification scanner. This practical application directly translates the theoretical advantages of ESA-FPM into transformative benefits for pathological workflow:

• Dramatically Increased Scanning Speed: By requiring only 7 images instead of hundreds to synthesize a high-resolution, large-FOV image, the scanner leverages ESA-FPM’s efficiency to achieve scan times orders of magnitude faster than traditional high-resolution methods. This enables rapid whole-slide digitization, crucial for high-volume diagnostic laboratories.

• Elimination of Oil-Immersion Procedure: Conventional high-resolution (100x) microscopy relies on oil-immersion objective lenses to achieve high numerical aperture (NA), necessitating a messy and time-consuming process of applying and cleaning immersion oil. ESA-FPM fundamentally bypasses this need. It uses a low-magnification, high-NA dry objective lens and computationally synthesizes the equivalent of a much higher NA. This allows the KFBIO scanner to deliver oil-immersion-level resolution without the oil, streamlining operations, reducing costs, and improving workflow reliability.

This successful integration of ESA-FPM into a commercial-grade scanner stands as a testament to the powerful synergy between KFBIO’s engineering expertise and SCI Lab’s computational imaging innovation, delivering a tangible solution that enhances both the speed and simplicity of digital pathology.

Research Foundation
This article presents applications of the ESA-FPM technology, a breakthrough developed jointly by KFBIO and Nanjing University of Science and Technology’s SCI Lab. The foundational research is published in:
Fan et al., Laser & Photonics Rev. 2022, 2200201. https://doi.org/10.1002/lpor.202200201