We present a new label-free three-dimensional (3D) microscopy technique, termed transport of intensity diffraction tomography with non-interferometric synthetic aperture (TIDT-NSA). Without resorting to interferometric detection, TIDT-NSA retrieves the 3D refractive index (RI) distribution of biological specimens from 3D intensity-only measurements at various illumination angles, allowing incoherent-diffraction-limited quantitative 3D phase-contrast imaging. The unique combination of z-scanning the sample with illumination angle diversity in TIDT-NSA provides strong defocus phase contrast and better optical sectioning capabilities suitable for high-resolution tomography of thick biological samples. Based on an off-the-shelf bright-field microscope with a programmable light-emitting-diode (LED) illumination source, TIDT-NSA achieves an imaging resolution of 206 nm laterally and 520 nm axially with a high-NA oil immersion objective. We validate the 3D RI tomographic imaging performance on various unlabeled fixed and live samples, including human breast cancer cell lines MCF-7, human hepatocyte carcinoma cell lines HepG2, mouse macrophage cell lines RAW 264.7, Caenorhabditis elegans (C. elegans), and live Henrietta Lacks (HeLa) cells. These results establish TIDT-NSA as a new non-interferometric approach to optical diffraction tomography and 3D label-free microscopy, permitting quantitative characterization of cell morphology and time-dependent subcellular changes for widespread biological and medical applications.

Quantitative phase imaging (QPI) has emerged as a valuable tool for biomedical research thanks to its unique capabilities for quantifying optical thickness variation of living cells and tissues. Among many QPI methods, Fourier ptychographic microscopy (FPM) allows long-term label-free observation and quantitative analysis of large cell populations without compromising spatial and temporal resolution. However, high spatio-temporal resolution imaging over a long-time scale (from hours to days) remains a critical challenge: optically inhomogeneous structure of biological specimens as well as mechanical perturbations and thermal fluctuations of the microscope body all result in time-varying aberration and focus drifts, significantly degrading the imaging performance for long-term study. Moreover, the aberrations are sample- and environment-dependent, and cannot be compensated by a fixed optical design, thus necessitating rapid dynamic correction in the imaging process. Here, we report an adaptive optical QPI method based on annular illumination FPM. In this method, the annular matched illumination configuration (i.e., the illumination numerical aperture (NA) strictly equals to the objective NA), which is the key for recovering low-frequency phase information, is further utilized for the accurate imaging aberration characterization. By using only 6 low-resolution images captured with 6 different illumination angles matching the NA of a 10x, 0.4 NA objective, we recover high-resolution quantitative phase images (synthetic NA of 0.8) and characterize the aberrations in real time, restoring the optimum resolution of the system adaptively. Applying our method to live-cell imaging, we achieve diffraction-limited performance (full-pitch resolution of $$655\,nm$$ 655 n m at a wavelength of $$525\,nm$$ 525 n m ) across a wide field of view ($$1.77\,mm^2$$ 1.77 m m 2 ) over an extended period of time.

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Optical diffraction tomography (ODT) is a promising label-free three-dimensional (3D) microscopic method capable of measuring the 3D refractive index (RI) distribution of optically transparent samples (e.g., unlabeled biological cells). In recent years, non-interferometric ODT techniques have received increasing attention for their system simplicity, speckle-free imaging quality, and compatibility with existing microscopes. However, ODT methods for implementing non-interferometric measurements in high numerical aperture (NA) microscopy systems are often plagued by low-frequency missing problems—a consequence of violating the matched illumination condition. Here, we present transport-of-intensity Fourier ptychographic diffraction tomography (TI-FPDT) to address this challenging issue by combining ptychographic angular diversity with additional “transport of intensity” measurements. TI-FPDT exploits the defocused phase contrast to circumvent the stringent requirement on the illumination NA imposed by the matched illumination condition. It effectively overcomes the reconstruction quality deterioration and RI underestimation problems in conventional FPDT, as demonstrated by high-resolution tomographic imaging of various unlabeled transparent samples (including microspheres, USAF targets, HeLa cells, and C2C12 cells). Due to its simplicity and effectiveness, TI-FPDT is anticipated to open new possibilities for label-free 3D microscopy in various biomedical applications.

Fourier ptychographic diffraction tomography (FPDT) is a recently developed label‐free computational microscopy technique that retrieves high‐resolution and large‐field three‐dimensional (3D) tomograms by synthesizing a set of low‐resolution intensity images obtained with a low numerical aperture (NA) objective. However, in order to ensure sufficient overlap of Ewald spheres in 3D Fourier space, conventional FPDT requires thousands of intensity measurements and consumes a significant amount of time for stable convergence of the iterative algorithm. Herein, we present accelerated Fourier ptychographic diffraction tomography (aFPDT), which combines sparse annular light‐emitting diode (LED) illuminations and multiplexing illumination to significantly decrease data amount and achieve computational acceleration of 3D refractive index (RI) tomography. Compared with existing FPDT technique, the equivalent high‐resolution 3D RI results are obtained using aFPDT with reducing data requirement by more than 40 times. The validity of the proposed method is experimentally demonstrated on control samples and various biological cells, including polystyrene beads, unicellular algae and clustered HeLa cells in a large field of view. With the capability of high‐resolution and high‐throughput 3D imaging using small amounts of data, aFPDT has the potential to further advance its widespread applications in biomedicine.