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Imaging flow cytometry (IFC) combines flow cytometry and fluorescence microscopy to enable high-throughput, multiparametric single-cell analysis with rich spatial details. However, current IFC techniques remain limited in their ability to reveal subcellular information with a high 3D resolution, throughput, sensitivity, and instrumental simplicity. In this study, we introduce a light-field flow cytometer (LFC), an IFC system capable of high-content, single-shot, and multi-color acquisition of up to 5,750 cells per second with a near-diffraction-limited resolution of 400-600 nm in all three dimensions. The LFC system integrates optical, microfluidic, and computational strategies to facilitate the volumetric visualization of various 3D subcellular characteristics through convenient access to commonly used epi-fluorescence platforms. We demonstrate the effectiveness of LFC in assaying, analyzing, and enumerating intricate subcellular morphology, function, and heterogeneity using various phantoms and biological specimens. The advancement offered by the LFC system presents a promising methodological pathway for broad cell biological and translational discoveries, with the potential for widespread adoption in biomedical research.

Fluorescence microscopy is one of the most indispensable and informative driving forces for biological research, but the extent of observable biological phenomena is essentially determined by the content and quality of the acquired images. To address the different noise sources that can degrade these images, we introduce an algorithm for multiscale image restoration through optimally sparse representation (MIRO). MIRO is a deterministic framework that models the acquisition process and uses pixelwise noise correction to improve image quality. Our study demonstrates that this approach yields a remarkable restoration of the fluorescence signal for a wide range of microscopy systems, regardless of the detector used (e.g., electron-multiplying charge-coupled device, scientific complementary metal-oxide semiconductor, or photomultiplier tube). MIRO improves current imaging capabilities, enabling fast, low-light optical microscopy, accurate image analysis, and robust machine intelligence when integrated with deep neural networks. This expands the range of biological knowledge that can be obtained from fluorescence microscopy.

This study introduces a rapid, volumetric live-cell imaging technique for visualizing autofluorescent sub-cellular structures and their dynamics by employing high-resolution Fourier light-field microscopy. We demonstrated this method by capturing lysosomal autofluorescence in fibroblasts and HeLa cells. Additionally, we conducted multicolor imaging to simultaneously observe lysosomal autofluorescence and fluorescently-labeled organelles such as lysosomes and mitochondria. We further analyzed the data to quantify the interactions between lysosomes and mitochondria. This research lays the foundation for future exploration of native cellular states and functions in three-dimensional environments, effectively reducing photodamage and eliminating the necessity for exogenous labels.

Characterizing the structural properties of galaxies in high-redshift protoclusters is key to our understanding of the environmental effects on galaxy evolution in the early stages of galaxy and structure formation. In this study, we assess the structural properties of 85 and 87 Hα emission-line candidates (HAEs) in the densest regions of two massive protoclusters, BOSS1244 and BOSS1542, respectively, using the Hubble Space Telescope (HST) H-band imaging data. Our results show a true pair fraction of 22 ± 5 (33 ± 6) per cent in BOSS1244 (BOSS1542), which yields a merger rate of 0.41 ± 0.09 (0.52 ± 0.04) Gyr−1 for massive HAEs with log (M*/M⊙) ≥ 10.3. This rate is 1.8 (2.8) times higher than that of the general fields at the same epoch. Our sample of HAEs exhibits half-light radii and Sérsic indices that cover a broader range than field star-forming galaxies. Additionally, about 15 per cent of the HAEs are as compact as the most massive (log (M*/M⊙) ≳ 11) spheroid-dominated population. These results suggest that the high galaxy density and cold dynamical state (i.e. velocity dispersion of <400 km s−1) are key factors that drive galaxy mergers and promote structural evolution in the two protoclusters. Our findings also indicate that both the local environment (on group scales) and the global environment play essential roles in shaping galaxy morphologies in protoclusters. This is evident in the systematic differences observed in the structural properties of galaxies between BOSS1244 and BOSS1542.

Live-cell imaging reveals the phenotypes and mechanisms of cellular function and their dysfunction that underscore cell physiology, development, and pathology. Here, we report a 3D super-resolution live-cell microscopy method by integrating radiality analysis and Fourier light-field microscopy (rad-FLFM). We demonstrated the method using various live-cell specimens, including actins in Hela cells, microtubules in mammary organoid cells, and peroxisomes in COS-7 cells. Compared with conventional wide-field microscopy,rad-FLFM realizes scanning-free, volumetric 3D live-cell imaging with sub-diffraction-limited resolution of ∼150 nm (x-y) and 300 nm (z), milliseconds volume acquisition time, six-fold extended depth of focus of ∼6 µm, and low photodamage. The method provides a promising avenue to explore spatiotemporal-challenging subcellular processes in a wide range of cell biological research.

Volumetric interrogation of the organization and processes of intracellular organelles and molecules in cellular systems with a high spatiotemporal resolution is essential for understanding cell physiology, development, and pathology. Here, we report high-resolution Fourier light-field microscopy (HR-FLFM) for fast and volumetric live-cell imaging. HR-FLFM transforms conventional cell microscopy and enables exploration of less accessible spatiotemporal-limiting regimes for single-cell studies. The results present a near-diffraction-limited resolution in all three dimensions, a five-fold extended focal depth to several micrometers, and a scanning-free volume acquisition time up to milliseconds. The system demonstrates instrumentation accessibility, low photo damage for continuous observation, and high compatibility with general cell assays. We anticipate HR-FLFM to offer a promising methodological pathway for investigating a wide range of intracellular processes and functions with exquisite spatiotemporal contextual details.