Fourier light-field imaging of human organoids with a hybrid point-spread function.

Volumetric interrogation of the cellular morphology and dynamic processes of organoid systems with a high spatiotemporal resolution provides critical insights for understanding organogenesis, tissue homeostasis, and organ function. Fluorescence microscopy has emerged as one of the most vital and informative driving forces for probing the cellular complexity in organoid research. However, the underlying scanning mechanism of conventional imaging methods inevitably compromises the time resolution of volumetric acquisition, leading to increased photodamage and inability to capture fast cellular and tissue dynamic processes. Here, we report Fourier light-field microscopy using a hybrid point-spread function (hPSF-FLFM) for fast, volumetric, and high-resolution imaging of entire organoids. hPSF-FLFM transforms conventional 3D microscopy and enables exploration of less accessible spatiotemporally-challenging regimes for organoid research. To validate hPSF-FLFM, we demonstrate 3D imaging of rapid responses to extracellular physical cues such as osmotic and mechanical stresses on human induced pluripotent stem cells-derived colon organoids (hCOs). The system offers cellular (2-3 µm and 5-6 µm in x-y and z, respectively) and millisecond-scale spatiotemporal characterization of whole-organoid dynamic changes that span large imaging volumes (>900 µm × 900 µm × 200 µm in x, y, z, respectively). The hPSF-FLFM method provides a promising avenue to explore spatiotemporal-challenging cellular responses in a wide variety of organoid research.

Perfusion System for Modification of Luminal Contents of Human Intestinal Organoids and Realtime Imaging Analysis of Microbial Populations.

Intestinal organoids are 3D cell structures that replicate some aspects of organ function and are organized with a polarized epithelium facing a central lumen. To enable more applications, new technologies are needed to access the luminal cavity and apical cell surface of organoids. We developed a perfusion system utilizing a double-barrel glass capillary with a pressure-based pump to access and modify the luminal contents of a human intestinal organoid for extended periods of time while applying cyclic cellular strain. Cyclic injection and withdrawal of fluorescent FITC-Dextran coupled with real-time measurement of fluorescence intensity showed discrete changes of intensity correlating with perfusion cycles. The perfusion system was also used to modify the lumen of organoids injected with GFP-expressing E. coli. Due to the low concentration and fluorescence of the E. coli, a novel imaging analysis method utilizing bacteria enumeration and image flattening was developed to monitor E. coli within the organoid. Collectively, this work shows that a double-barrel perfusion system provides constant luminal access and allows regulation of luminal contents and luminal mixing.

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.

Protocell arrays for simultaneous detection of diverse analytes.

Simultaneous detection of multiple analytes from a single sample (multiplexing), particularly when done at the point of need, can guide complex decision-making without increasing the required sample volume or cost per test. Despite recent advances, multiplexed analyte sensing still typically faces the critical limitation of measuring only one type of molecule (e.g., small molecules or nucleic acids) per assay platform. Here, we address this bottleneck with a customizable platform that integrates cell-free expression (CFE) with a polymer-based aqueous two-phase system (ATPS), producing membrane-less protocells containing transcription and translation machinery used for detection. We show that multiple protocells, each performing a distinct sensing reaction, can be arrayed in the same microwell to detect chemically diverse targets from the same sample. Furthermore, these protocell arrays are compatible with human biofluids, maintain function after lyophilization and rehydration, and can produce visually interpretable readouts, illustrating this platform's potential as a minimal-equipment, field-deployable, multi-analyte detection tool.

Integration of Sensors in Gastrointestinal Organoid Culture for Biological Analysis.

The gastrointestinal (GI) tract regulates physiologic responses in complex ways beyond facilitating nutrient entry into the circulatory system. Because of the anatomic location of the GI tract, studying in vivo physiology of the human gut, including host cell interaction with the microbiota, is limited. GI organoids derived from human stem cells are gaining interest as they recapitulate in vivo cellular phenotypes and functions. An underdeveloped capability that would further enhance the utility of these miniature models of the GI tract is to use sensors to quantitatively characterize the organoid systems with high spatiotemporal resolution. In this review, we first discuss tools to capture changes in the fluid milieu of organoid cultures both in the organoid exterior as well as the luminal side of the organoids. The subsequent section describes approaches to characterize barrier functions across the epithelial layer of the GI organoids directly or after transferring the epithelial cells to a 2-dimensional culture format in Transwells or compartmentalized microchannel devices. The final section introduces recently developed bioengineered bacterial sensors that sense intestinal inflammation-related small molecules in the lumen using lambda cI/Cro genetic elements or fluorescence as readouts. Considering the small size and cystic shape of GI organoids, sensors used in conventional macroscopic intestinal models are often not suitable, particularly for time-lapse monitoring. Unmet needs for GI organoid analysis provides many opportunities for the development of noninvasive and miniaturized biosensors.

Dispersible oxygen microsensors map oxygen gradients in three-dimensional cell cultures.

Phase fluorimetry, unlike the more commonly used intensity-based measurement, is not affected by differences in light paths from culture vessels or by optical attenuation through dense 3D cell cultures and hydrogels thereby minimizing dependence on signal intensity for accurate measurements. This work describes the use of phase fluorimetry on oxygen-sensor microbeads to perform oxygen measurements in different microtissue culture environments. In one example, cell spheroids were observed to deplete oxygen from the cell-culture medium filling the bottom of conventional microwells within minutes, whereas oxygen concentrations remained close to ambient levels for several days in hanging-drop cultures. By dispersing multiple oxygen microsensors in cell-laden hydrogels, we also mapped cell-generated oxygen gradients. The spatial oxygen mapping was sufficiently precise to enable the use of computational models of oxygen diffusion and uptake to give estimates of the cellular oxygen uptake rate and the half-saturation constant. The results show the importance of integrated design and analysis of 3D cell cultures from both biomaterial and oxygen supply aspects. While this paper specifically tests spheroids and cell-laden gel cultures, the described methods should be useful for measuring pericellular oxygen concentrations in a variety of biomaterials and culture formats.

Bioengineering for intestinal organoid cultures.

Recent advances allow access to human cell-based intestinal organoids that recreate human physiology to levels not possible with conventional 2D cell cultures. Despite their huge potential, there are many challenges that remain. This review will cover recent bioengineering approaches to improve organoid maturation, scale up, reproducibility and analysis. The first section covers the advances in engineering the culture environment, followed by the section on tools for micro-manipulation and analysis of organoids. The last section reviews the computational models developed to guide the use of engineered materials and tools, and to interpret observed results as well. The ability to use organoids for discovery research, and the need to both exert exquisite experimental control and obtain quantitative measurements from organoid models means that the field is ripe for collaborative efforts between biologists, engineers, clinicians and industry.