hydroSIM: super-resolution speckle illumination microscopy with a hydrogel diffuser.

Super-resolution microscopy has emerged as an indispensable methodology for probing the intricacies of cellular biology. Structured illumination microscopy (SIM), in particular, offers an advantageous balance of spatial and temporal resolution, allowing for visualizing cellular processes with minimal disruption to biological specimens. However, the broader adoption of SIM remains hampered by the complexity of instrumentation and alignment. Here, we introduce speckle-illumination super-resolution microscopy using hydrogel diffusers (hydroSIM). The study utilizes the high scattering and optical transmissive properties of hydrogel materials and realizes a remarkably simplified approach to plug-in super-resolution imaging via a common epi-fluorescence platform. We demonstrate the hydroSIM system using various phantom and biological samples, and the results exhibited effective 3D resolution doubling, optical sectioning, and high contrast. We foresee hydroSIM, a cost-effective, biocompatible, and user-accessible super-resolution methodology, to significantly advance a wide range of biomedical imaging and applications.

Light-field flow cytometry for high-resolution, volumetric and multiparametric 3D single-cell analysis.

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.

Cationic cholesterol-dependent LNP delivery to lung stem cells, the liver, and heart.

Adding a cationic helper lipid to a lipid nanoparticle (LNP) can increase lung delivery and decrease liver delivery. However, it remains unclear whether charge-dependent tropism is universal or, alternatively, whether it depends on the component that is charged. Here, we report evidence that cationic cholesterol-dependent tropism can differ from cationic helper lipid-dependent tropism. By testing how 196 LNPs delivered mRNA to 22 cell types, we found that charged cholesterols led to a different lung:liver delivery ratio than charged helper lipids. We also found that combining cationic cholesterol with a cationic helper lipid led to mRNA delivery in the heart as well as several lung cell types, including stem cell-like populations. These data highlight the utility of exploring charge-dependent LNP tropism.

Three-dimensional multifocal scanning microscopy for super-resolution cell and tissue imaging.

Recent advancements in image-scanning microscopy have significantly enriched super-resolution biological research, providing deeper insights into cellular structures and processes. However, current image-scanning techniques often require complex instrumentation and alignment, constraining their broader applicability in cell biological discovery and convenient, cost-effective integration into commonly used frameworks like epi-fluorescence microscopes. Here, we introduce three-dimensional multifocal scanning microscopy (3D-MSM) for super-resolution imaging of cells and tissue with substantially reduced instrumental complexity. This method harnesses the inherent 3D movement of specimens to achieve stationary, multi-focal excitation and super-resolution microscopy through a standard epi-fluorescence platform. We validated the system using a range of phantom, single-cell, and tissue specimens. The combined strengths of structured illumination, confocal detection, and epi-fluorescence setup result in two-fold resolution improvement in all three dimensions, effective optical sectioning, scalable volume acquisition, and compatibility with general imaging and sample protocols. We anticipate that 3D-MSM will pave a promising path for future super-resolution investigations in cell and tissue biology.

Volumetric live-cell autofluorescence imaging using Fourier light-field 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.

Simultaneous Multicolor Multifocal Scanning Microscopy.

Super-resolution fluorescence microscopy has revolutionized cell biology over the past decade, enabling the visualization of subcellular complexity with unparalleled clarity and detail. However, the rapid development of image-scanning-based super-resolution systems still restrains convenient access to commonly used instruments such as epi-fluorescence microscopes. Here, we present multifocal scanning microscopy (MSM) for super-resolution imaging with simultaneous multicolor acquisition and minimal instrumental complexity. MSM implements a stationary, interposed multifocal multicolor excitation by exploiting the motion of the specimens, realizing super-resolution microscopy through a general epi-fluorescence platform without compromising the image-scanning mechanism or inducing complex instrument alignment. The system is demonstrated with various phantom and biological specimens, and the results present effective resolution doubling, optical sectioning, and contrast enhancement. We anticipate MSM, as a highly accessible and compatible super-resolution technique, to offer a promising methodological pathway for broad cell biological discoveries.

3D super-resolution live-cell imaging with radial symmetry and Fourier light-field microscopy.

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.

Extending the Near-Infrared Emission Range of Indium Phosphide Quantum Dots for Multiplexed In Vivo Imaging.

This report of the reddest emitting indium phosphide quantum dots (InP QDs) to date demonstrates tunable, near-infrared (NIR) photoluminescence (PL) as well as PL multiplexing in the first optical tissue window while avoiding toxic constituents. This synthesis overcomes the InP "growth bottleneck" and extends the emission peak of InP QDs deeper into the first optical tissue window using an inverted QD heterostructure, specifically ZnSe/InP/ZnS core/shell/shell nanoparticles. The QDs exhibit InP shell thickness-dependent tunable emission with peaks ranging from 515-845 nm. The high absorptivity of InP yields effective photoexcitation of the QDs with UV, visible, and NIR wavelengths. These nanoparticles extend the range of tunable direct-bandgap emission from InP-based nanostructures, effectively overcoming a synthetic barrier that has prevented InP-based QDs from reaching their full potential as NIR imaging agents. Multiplexed lymph node imaging in a mouse model demonstrates the potential of the NIR-emitting InP particles for in vivo imaging.