Jiabei Zhu

Three-dimensional molecular imaging of living cells is essential for unraveling cellular metabolism and response to therapies. However, existing volumetric methods, including fluorescence microscopy and quantitative phase imaging, either require fluorescent labels or lack chemical specificity. Mid-infrared (mid-IR) photothermal microscopy provides label-free spectroscopic contrast with sub-micrometer resolution but is limited by slow acquisition rates, precluding 3D live-cell studies. Here, we present a photothermal relaxation intensity diffraction tomography (PRIDT) system that encodes mid-IR absorption-induced refractive index changes via a photothermal relaxation scheme and recovers it through intensity diffraction tomography. PRIDT achieves video-rate volumetric chemical imaging with up to 15 Hz per wavelength and offers lateral and axial resolutions of 264 nm and 1 . 12 µm, respectively, within a volumetric field of view of 50 × 50 × 10 µm ³ . We showcase high-speed PRIDT imaging of protein and lipid metabolism in ovarian cancer cells and lipid-droplet dynamics in live cells. PRIDT opens new avenues for rapid, quantitative, three-dimensional molecular imaging in living systems.

Diffraction tomography (DT) has been widely explored in transmission-mode configurations, enabling high-resolution, label-free 3D imaging. However, industrial metrology applications, such as semiconductor inspection, typically involve opaque or highly reflective substrates (e.g., silicon or metal), necessitating a reflection-mode imaging configuration. In this work, we introduce reflection-mode Multi-Slice Fourier Ptychographic Tomography (rMS-FPT) that achieves high-resolution, volumetric imaging of multi-layered, strongly scattering samples on reflective substrates. We develop a reflection-mode multi-slice beam propagation method (rMSBP) to model multiple scattering and substrate interactions, enabling precise 3D reconstruction. By incorporating darkfield measurements, rMS-FPT enhances resolution beyond the traditional brightfield limit and provides sub-micrometer lateral resolution while achieving optical sectioning. We validate rMS-FPT through numerical simulations on a four-layer resolution target and experimental demonstrations using a reflection-mode LED array microscope. Experiments on a two-layer resolution target and a multi-layer scattering sample confirm the method's effectiveness. Our optimized implementation enables rapid imaging, covering a 1.2mm × 1.2mm area in 1.6 seconds, reconstructing over 10⁹ voxels within a 0.4mm³ volume. This work represents a significant step in extending DT to reflection-mode configurations, providing a robust and scalable solution for 3D metrology and industrial inspection.

We introduce a reflection-mode diffraction tomography technique that enables the simultaneous recovery of forward- and backward-scattering information for high-resolution 3D refractive index reconstruction. Our technique works by imaging a sample on a highly reflective substrate and employing a multiple-scattering model and a reconstruction algorithm. It combines the modified Born series as the forward model, Bloch and perfect electric conductor boundary conditions to handle oblique incidence and substrate reflections, and the adjoint method for efficient gradient computation in solving the inverse-scattering problem. We validate the technique through simulations and experiments, achieving accurate reconstructions in samples with high refractive index contrasts and complex geometries. Forward scattering captures smooth axial features, while backward scattering reveals complementary interfacial details. Experimental results on dual-layer resolution targets, 3D randomly distributed beads, phase structures obscured by highly scattering fibers, fixed breast cancer cells, and fixed C. elegans demonstrate its robustness and versatility. This technique holds promise for applications in semiconductor metrology and biomedical imaging.

HiLo microscopy is an optical sectioning structured illumination microscopy technique based on computationally combining two images: one with uniform illumination and the other with structured illumination. The most widely used structured illumination in HiLo microscopy is random speckle patterns, due to their simplicity and resilience to tissue scattering. Here, we present a novel HiLo microscopy strategy based on random caustic patterns. Building on an off-the-shelf diffuser and a low-coherence LED source, we demonstrate that caustic HiLo can achieve 4.5 µm optical sectioning capability with a 20× 0.75 NA objective. In addition, with the distinct intensity statistical properties of caustic patterns, we show that our caustic HiLo outperforms speckle HiLo, achieving enhanced optical sectioning capability and preservation of fine features by imaging scattering fixed brain sections of 100 µm, 300 µm, and 500 µm thicknesses. We anticipate that this new structured illumination technique may find various biomedical imaging applications.

Topography measurement is essential for surface characterization, semiconductor metrology, and inspection applications. To date, performing high-throughput and accurate topography remains challenging due to the trade-off between field-of-view (FOV) and spatial resolution. Here we demonstrate a novel topography technique based on the reflection-mode Fourier ptychographic microscopy, termed Fourier ptychograhpic topography (FPT). We show that FPT provides both a wide FOV and high resolution, and achieves nanoscale height reconstruction accuracy. Our FPT prototype is based on a custom-built computational microscope consisting of programmable brightfield and darkfield LED arrays. The topography reconstruction is performed by a sequential Gauss-Newton-based Fourier ptychographic phase retrieval algorithm augmented with total variation regularization. We achieve a synthetic numerical aperture (NA) of 0.84 and a diffraction-limited resolution of 750 nm, increasing the native objective NA (0.28) by 3×, across a 1.2 × 1.2 mm² FOV. We experimentally demonstrate the FPT on a variety of reflective samples with different patterned structures. The reconstructed resolution is validated on both amplitude and phase resolution test features. The accuracy of the reconstructed surface profile is benchmarked against high-resolution optical profilometry measurements. In addition, we show that the FPT provides robust surface profile reconstructions even on complex patterns with fine features that cannot be reliably measured by the standard optical profilometer. The spatial and temporal noise of our FPT system is characterized to be 0.529 nm and 0.027 nm, respectively.

Recovering 3D phase features of complex biological samples traditionally sacrifices computational efficiency and processing time for physical model accuracy and reconstruction quality. Here, we overcome this challenge using an approximant-guided deep learning framework in a high-speed intensity diffraction tomography system. Applying a physics model simulator-based learning strategy trained entirely on natural image datasets, we show our network can robustly reconstruct complex 3D biological samples. To achieve highly efficient training and prediction, we implement a lightweight 2D network structure that utilizes a multi-channel input for encoding the axial information. We demonstrate this framework on experimental measurements of weakly scattering epithelial buccal cells and strongly scattering C. elegans worms. We benchmark the network’s performance against a state-of-the-art multiple-scattering model-based iterative reconstruction algorithm. We highlight the network’s robustness by reconstructing dynamic samples from a living worm video. We further emphasize the network’s generalization capabilities by recovering algae samples imaged from different experimental setups. To assess the prediction quality, we develop a quantitative evaluation metric to show that our predictions are consistent with both multiple-scattering physics and experimental measurements.

We demonstrate a novel topography technique based on the reflection-mode Fourier ptychographic microscopy, termed Fourier ptychograhpic topography (FPT), to provide a wide field-of-view, high resolution, and nanoscale topography reconstruction accuracy.

We propose a novel algorithm based on the split-step non-paraxial model for different intensity diffraction tomography setups to recover the 3D refractive index distribution of multiple-scattering biological samples.

We propose a novel intensity diffraction tomography (IDT) reconstruction algorithm based on the split-step non-paraxial (SSNP) model for recovering the 3D refractive index (RI) distribution of multiple-scattering biological samples. High-quality IDT reconstruction requires high-angle illumination to encode both low- and high- spatial frequency information of the 3D biological sample. We show that our SSNP model can more accurately compute multiple scattering from high-angle illumination compared to paraxial approximation-based multiple-scattering models. We apply this SSNP model to both sequential and multiplexed IDT techniques. We develop a unified reconstruction algorithm for both IDT modalities that is highly computationally efficient and is implemented by a modular automatic differentiation framework. We demonstrate the capability of our reconstruction algorithm on both weakly scattering buccal epithelial cells and strongly scattering live C. elegans worms and live C. elegans embryos.

Objective and Impact Statement Segmentation of blood vessels from two-photon microscopy (2PM) angiograms of brains has important applications in hemodynamic analysis and disease diagnosis. Here we develop a generalizable deep-learning technique for accurate 2PM vascular segmentation of sizable regions in mouse brains acquired from multiple 2PM setups. In addition, the technique is computationally efficient, making it ideal for large-scale neurovascular analysis. Introduction Vascular segmentation from 2PM angiograms is usually an important first step in hemodynamic modeling of brain vasculature. Existing state-of-the-art segmentation methods based on deep-learning either lack the ability to generalize to data from various imaging systems, or are computationally infeasible for large-scale angiograms. In this work, we present a method which improves upon both these limitations by being generalizable to various imaging systems, and also being able to segment very large-scale angiograms. Methods We employ a computationally efficient deep-learning framework based on a semi-supervised learning strategy, whose effectiveness we demonstrate on experimentally acquired in-vivo angiograms from mouse brains of dimensions up to 808×808×702 µm. Results After training on data from only one 2PM microscope, we perform vascular segmentation on data from another microscope without any network tuning. Our method demonstrates 10× faster computation in terms of voxels-segmented-per-second and 3× larger depth compared to the state-of-the-art. Conclusion Our work provides a generalizable and computationally efficient anatomical modeling framework for the brain vasculature, which consists of deep-learning based vascular segmentation followed by graphing. It paves the way for future modeling and analysis of hemodynamic response at much greater scales that were inaccessible before.