Computational label-free microscope through a custom-built high-throughput objective lens and Fourier ptychography

Abstract

Label-free microscopy directly images live samples without fluorescent markers, providing a panoramic view of biological structures and functions. However, pursuing high-throughput and high-content live cell imaging requires both higher spatial resolution and larger field of view, which are limited by the optical system. In this study, we custom-built a 5▪/0.35 objective with a field number of 28 mm, providing a space-bandwidth product of 34 megapixels, which is four times greater than that of commercial high-throughput objectives such as the Nikon Lambda series (4▪/0.2, 10▪/0.45, 20▪/0.75). Furthermore, we incorporated Fourier ptychography microscopy (FPM) into the system, achieving a synthetic numerical aperture of 0.72, suitable for digital pathology and long-term live cell observation. To improve the FPM performance, we proposed a phantom-based calibration method for quantitative correction of illumination angle errors. Additionally, this method can also serve as an initial step for the on-line calibration. The remarkable capabilities of our 5▪/0.35 objective have been demonstrated, as well as the effectiveness of computational label-free microscopy. Furthermore, by combining our original FPM with more advanced hardware and algorithms, our FPM can achieve higher performance.