A Custom-Built SPIM Platform for Three-Dimensional Time-Lapse Imaging and Quantification of Anisotropic Tumor Spheroid Growth

The three-dimensional (3D) tumor spheroid model is a critical tool for high-throughput ovarian cancer research and anticancer drug development in vitro. However, the 3D structure prevents high-resolution imaging of the inner side of the spheroids. We aim to visualize and characterize 3D morphological and physiological information of the contact multicellular ovarian tumor spheroids growing over time. We intend to further evaluate the distinctive evolutions of the tumor spheroid and necrotic tissue volumes in different cell numbers and determine the most appropriate mathematical model for fitting the growth of tumor spheroids and necrotic tissues. A label-free and noninvasive swept-source optical coherence tomography (SS-OCT) imaging platform was applied to obtain two-dimensional (2D) and 3D morphologies of ovarian tumor spheroids over 18 days. Ovarian tumor spheroids of two different initial cell numbers (5,000- and 50,000- cells) were cultured and imaged (each day) over the time of growth in 18 days. Four mathematical models (Exponential-Linear, Gompertz, logistic, and Boltzmann) were employed to describe the growth kinetics of the tumor spheroids volume and necrotic tissues. Ovarian tumor spheroids have different growth curves with different initial cell numbers and their growths contain different stages with various growth rates over 18 days. The volumes of 50,000-cells spheroids and the corresponding necrotic tissues are larger than that of the 5,000-cells spheroids. The formation of necrotic tissue in 5,000-cells numbers is slower than that in the 50,000-cells ones. Moreover, the Boltzmann model exhibits the best fitting performance for the growth of tumor spheroids and necrotic tissues. Optical coherence tomography (OCT) can serve as a promising imaging modality to visualize and characterize morphological and physiological features of multicellular ovarian tumor spheroids. The Boltzmann model integrating with 3D OCT data of ovarian tumor spheroids provides great potential for high-throughput cancer research in vitro and aiding in drug development.

Three-Dimensional Cross-Sectional Light-Sheet Microscopy Imaging of the Inflamed Mouse Gut

Endogenous electrical fields play an important role in various physiological and pathological events. Yet the effects of electrical cues on processes such as wound healing, tumor development or metastasis are still rarely investigated, though it is known that direct current electrical fields can alter cell migration or proliferation in vitro. Several 2D experimental models for studying cell responses to direct current electrical fields have been presented and characterized but suitable experimental models for electrotaxis studies in 3D are rare. Here we present a novel, easy-to-produce, multi-well-based galvanotactic-chamber for the use in 2D and 3D cell experiments for investigations on the influence of electrical fields on tumor cell migration and tumor spheroid growth. Our presented system allows the simultaneous application of electrical field to cells in four chambers, either cultured on the bottom of the culture-plate (2D) or embedded in hydrogel filled channels (3D). The set-up is also suitable for, live-cell-imaging. Validation tests show stable electrical fields and high cell viabilities inside the channel. Tumor spheroids of various diameters can be exposed to direct current electrical fields up to one week.

Objective: This study aimed to systematically evaluate the effects of human cytokines on the growth and transcriptional regulation of pancreatic ductal adenocarcinoma (PDAC) cells, using 3D tumor spheroid models to identify key cytokine–gene interactions relevant to tumor progression. Methods: Human PDAC cell lines (PANC-1, CFPAC-1) were cultured in Matrigel-based 3D tumor spheroid systems and treated with a panel of 365 human cytokines. Tumor spheroid number and size were quantified over time. RNA sequencing and quantitative polymerase chain reaction (PCR) were performed to profile transcriptional changes induced by transforming growth factor beta 1 (TGFB1) in 2D versus 3D cultures. Peptidyl arginine deiminase 2 (PADI2) function was validated using short hairpin RNA (shRNA)-mediated knockdown, followed by growth assays and western blot analysis. Results: TGFB1 suppressed pancreatic tumor spheroid formation and growth in 3D culture but showed no effect in 2D monolayer systems. Transcriptomic profiling revealed distinct TGFB1-regulated pathways in 3D spheroids, including phosphoinositide 3-kinase (PI3K)–protein kinase B (AKT), wingless and int-1 (WNT), and amebiasis signaling, while proteoglycan and focal adhesion pathways predominated in 2D. PADI2 expression was selectively upregulated by TGFB1 in 3D spheroids but not in 2D cultures. Functionally, PADI2 knockdown inhibited 3D tumor spheroid growth and potentiated TGFB1-mediated suppression, indicating that PADI2 supports tumor cell survival under TGFB1-induced stress. Conclusions: 3D tumor spheroid models better capture physiologically relevant cytokine responses than conventional 2D systems. TGFB1 acts as a potent inhibitor of pancreatic tumor spheroid growth through model-specific transcriptional reprogramming, while PADI2 serves as a TGFB1-induced survival factor and potential therapeutic target in PDAC.

Assessment of preclinical models of vascular disease is paramount in the successful translation of novel treatments. The results of these models have traditionally relied on two-dimensional (2D) histological methodologies. Light sheet fluorescence microscopy (LSFM) is an imaging platform that allows for three-dimensional (3D) visualization of whole organs and tissues. In this study, we describe an improved methodological approach utilizing LSFM for imaging of preclinical vascular injury models while minimizing analysis bias. The rat carotid artery segmental pressure-controlled balloon injury and mouse carotid artery ligation injury were performed. Arteries were harvested and processed for LSFM imaging and 3D analysis, as well as for 2D area histological analysis. Artery processing for LSFM imaging did not induce vessel shrinkage or expansion and was reversible by rehydrating the artery, allowing for subsequent sectioning and histological staining a posteriori. By generating a volumetric visualization along the length of the arteries, LSFM imaging provided different analysis modalities including volumetric, area, and radial parameters. Thus, LSFM-imaged arteries provided more precise measurements compared to classic histological analysis. Furthermore, LSFM provided additional information as compared to 2D analysis in demonstrating remodelling of the arterial media in regions of hyperplasia and periadventitial neovascularization around the ligated mouse artery. LSFM provides a novel and robust 3D imaging platform for visualizing and quantifying arterial injury in preclinical models. When compared with classic histology, LSFM outperformed traditional methods in precision and quantitative capabilities. LSFM allows for more comprehensive quantitation as compared to traditional histological methodologies, while minimizing user bias associated with area analysis of alternating, 2D histological artery cross-sections.

SUMMARYThe evaluation of DNA damage response, particularly DNA damage foci formation, is crucial for understanding tumor biology and assessing the impacts of various drugs. We have developed a sophisticated semi-automated image analysis pipeline which generates quantitative map of the spatiotemporal distribution of DNA damage foci within live tumor spheroids. Our framework seamlessly integrates live imaging of tumor spheroids via Light Sheet Fluorescence Microscopy with a DNA damage foci formation assay using a genetically encoded fluorescently labeled DNA damage sensor. By combining advanced imaging techniques with computational tools, our framework offers a powerful tool for studying DNA damage response mechanisms in complex 3D cellular environments.MOTIVATIONThe motivation of this work is to propose a comprehensive framework that facilitates the study of DNA repair mechanisms within 3D contexts, specifically using tumor spheroid models. By integrating advanced imaging technologies and genetically encoded fluorescent sensors, our goal is to offer researchers a robust methodology for observing and analyzing DNA damage dynamics in realistic tissue-like environments. This framework is designed to enhance accessibility and streamline data processing, thereby empowering the scientific community to investigate DNA repair processes in 3D with greater precision and efficiency.

Selective plane illumination microscopy (SPIM) is an optical sectioning technique that allows imaging of biological samples at high spatio-temporal resolution. Standard SPIM devices require dedicated set-ups, complex sample preparation and accurate system alignment, thus limiting the automation of the technique, its accessibility and throughput. We present a millimeter-scaled optofluidic device that incorporates selective plane illumination and fully automatic sample delivery and scanning. To this end an integrated cylindrical lens and a three-dimensional fluidic network were fabricated by femtosecond laser micromachining into a single glass chip. This device can upgrade any standard fluorescence microscope to a SPIM system. We used SPIM on a CHIP to automatically scan biological samples under a conventional microscope, without the need of any motorized stage: tissue spheroids expressing fluorescent proteins were flowed in the microchannel at constant speed and their sections were acquired while passing through the light sheet. We demonstrate high-throughput imaging of the entire sample volume (with a rate of 30 samples/min), segmentation and quantification in thick (100-300 μm diameter) cellular spheroids. This optofluidic device gives access to SPIM analyses to non-expert end-users, opening the way to automatic and fast screening of a high number of samples at subcellular resolution.

In order to determine the role of micromilieu in tumour spheroid growth, a mathematical model was developed to predict EMT6/Ro spheroid growth and microenvironment based upon numerical solution of the diffusion/reaction equation for oxygen, glucose, lactate ion, carbon dioxide, bicarbonate ion, chlorine ion and hydrogen ion along with the equation of electroneutrality. This model takes into account the effects of oxygen concentration, glucose concentration and extracellular pH on cell growth and metabolism. Since independent measurements of EMT6/Ro single cell growth and metabolic rates, spheroid diffusion constants, and spinner flask mass transfer coefficients are available, model predictions using these parameters were compared with published data on EMT6/Ro spheroid growth and micro-environment. The model predictions of reduced spheroid growth due to reduced cell growth rates and cell shedding fit experimental spheroid growth data below 700 microns, but overestimated the spheroid growth rate at larger diameters. Predicted viable rim thicknesses based on predicted near zero glucose concentrations fit published viable rim thickness data for 1000 microns spheroids grown at medium glucose concentrations of 5.5 mM or less. However, the model did not accurately predict the onset of necrosis. Moreover, the model could not predict the observed decreases in oxygen and glucose metabolism seen in spheroids with time, nor could it predict the observed growth plateau. This suggests that other unknown factors, such as inhibitors or cell-cell contact effects, must also be important in affecting spheroid growth and cellular metabolism.

Fluorescence Anisotropy Imaging in 3D with Single Plane Illumination Microscopy

Whole-brain imaging is important for understanding brain functions through deciphering tissue structures, neuronal circuits, and single-neuron tracing. Thus, many clearing methods have been developed to acquire whole-brain images or images of three-dimensional thick tissues. However, there are several limitations to imaging whole-brain volumes, including long image acquisition times, large volumes of data, and a long post-image process. Based on these limitations, many researchers are unsure about which light microscopy is most suitable for imaging thick tissues. Here, we compared fast-confocal microscopy with light-sheet fluorescence microscopy for whole-brain three-dimensional imaging, which can acquire images the fastest. To compare the two types of microscopies for large-volume imaging, we performed tissue clearing of a whole mouse brain, and changed the sample chamber and low- magnification objective lens and modified the sample holder of a light-sheet fluorescence microscope. We found out that light-sheet fluorescence microscopy using a 2.5× objective lens possesses several advantages, including saving time, large-volume image acquisitions, and high Z-resolution, over fast-confocal microscopy, which uses a 4× objective lens. Therefore, we suggest that light-sheet fluorescence microscopy is suitable for whole mouse brain imaging and for obtaining high-resolution three-dimensional images.

Selective Plane Illumination Microscopy in the Conventional Inverted Microscope Geometry

We describe a microscope capable of both light sheet fluorescence microscopy and differential interference contrast microscopy (DICM). The two imaging modes, which to the best of our knowledge have not previously been combined, are complementary: light sheet fluorescence microscopy provides three-dimensional imaging of fluorescently labelled components of multicellular systems with high speed, large fields of view, and low phototoxicity, whereas differential interference contrast microscopy reveals the unlabelled neighbourhood of tissues, organs, and other structures with high contrast and inherent optical sectioning. Use of a single Nomarski prism for differential interference contrast microscopy and a shared detection path for both imaging modes enables simple integration of the two techniques in one custom microscope. We provide several examples of the utility of the resulting instrument, focusing especially on the digestive tract of the larval zebrafish, revealing in this complex and heterogeneous environment anatomical features, the behaviour of commensal microbes, immune cell motions, and more.

.Significance: Selective plane illumination microscopy (SPIM) is an emerging fluorescent imaging technique suitable for noninvasive volumetric imaging of C. elegans. These promising microscopy systems, however, are scarce in academic and research institutions due to their high cost and technical complexities. Simple and low-cost solutions that enable conversion of commonplace wide-field microscopes to rapid SPIM platforms promote widespread adoption of SPIM by biologist for studying neuronal expressions of C. elegans.Aim: We sought to develop a simple and low-cost optofluidic add-on device that enables rapid and immobilization-free volumetric SPIM imaging of C. elegans with conventional fluorescent microscopes.Approach: A polydimethylsiloxane (PDMS)-based device with integrated optical and fluidic elements was developed as a low-cost and miniaturized SPIM add-on for the conventional wide-field microscope. The developed optofluidic chip contained an integrated PDMS cylindrical lens for on-chip generation of the light-sheet across a microchannel. Cross-sectional SPIM images of C. elegans were continuously acquired by the native objective of microscope as worms flowed in an L-shape microchannel and through the light sheet.Results: On-chip SPIM imaging of C. elegans strains demonstrated possibility of visualizing the entire neuronal system in few seconds at single-neuron resolution, with high contrast and without worm immobilization. Volumetric visualization of neuronal system from the acquired cross-sectional two-dimensional images is also demonstrated, enabling the standard microscope to acquire three-dimensional fluorescent images of C. elegans. The full-width at half-maximum width of the point spread function was measured as 1.1 and in the lateral and axial directions, respectively.Conclusion: The developed low-cost optofluidic device is capable of continuous SPIM imaging of C. elegans model organism with a conventional fluorescent microscope, at high speed, and with single neuron resolution.

For the investigation of three‐dimensional (3D) tumor spheroids and migrating cells embedded in a hydrogel using light sheet fluorescence microscopy (LSFM), a novel approach has been developed. LSFM has become a powerful tool for visualizing 3D structures of fluorescently labeled biological samples, providing high‐resolution imaging with minimal phototoxicity. Unlike conventional widefield or confocal microscopy methods, LSFM selectively illuminates a sample using a thin light sheet, thereby minimizing out‐of‐focus signals and facilitating quick volumetric data collection. However, sample mounting for LSFM still requires improvement, as conventional strategies, such as capillary‐based methods, present challenges especially for larger or delicate specimens, and may include mechanical stress. In this work, a novel sample holder compatible with the Zeiss Lightsheet 7 microscope is presented, specifically designed for the analysis of spheroids embedded in collagen gels. The novel holder enables dual‐sided illumination, improving effective image quality and facilitating detailed analysis of cell migration within the collagen matrix. Images acquired with this setup are analyzed using Arivis Vision4D to assess parameters such as cell number, cell density, and cell distance. The results demonstrated significant improvements not only in studying cancer cell migration and early metastatic behavior but also in evaluating chemotherapy within 3D extracellular matrix models.

Early-stage embryonic development involves highly dynamic and tightly regulated structural and molecular changes of tissues and cells. Capturing these critical events in time requires advanced imaging modalities that can provide simultaneous structural and molecular information. Therefore, significant effort has been devoted to developing imaging modalities capable of both structural and molecular imaging. However, many of these techniques cannot capture this multimodal information concurrently, resulting in the loss of critical dynamic information during embryogenesis. To address this challenge, we have developed a combined co-planar optical coherence tomography (OCT) and light-sheet fluorescence microscopy (LSFM) system, named OCT-LS, for long-term and multimodal embryonic imaging. OCT provides label-free structural information, while LSFM can acquire the corresponding molecular information concurrently. The system integrates both OCT and LSFM excitation beams through shared scan paths to ensure spatial co-registration. We validated the long-term imaging capability of OCT-LS by imaging transgenic zebrafish embryos Tg(kdrl:EGFP), where GFP-expressing vascular endothelial cells were visualized. This multimodality captured critical structural and molecular events such as brain, heart, and ocular development, all of which are resolved in real-time with high spatio-temporal fidelity. To further showcase the versatility of this system, we also utilized OCT-LS to investigate the teratogenic effect of prenatal ethanol exposure. Ethanol-treated (1.0%, 1.5%, and 2.0%) zebrafish embryos showed notable structural abnormalities, including reduced body length, eye diameter, and yolk sac volume compared to controls as captured by OCT. Concurrently, disruptions were observed in the caudal vessels, common cardinal veins, and somite segmentation patterns associated with ethanol-induced teratogenesis as captured by LSFM. Our results demonstrate that OCT-LS provides a powerful platform for long-term multimodal imaging of embryogenesis in small animals with broad applications in developmental biology, such as characterizing the spatio-temporal developmental defects associated with prenatal alcohol exposure.