Microscopy is a foundational tool for scientific exploration, allowing observation of biological structures. This visualization is instrumental in understanding life processes and disease mechanisms. However, conventional microscopy faces limitations in capturing the complexity of three-dimensional biological systems. Overcoming these challenges requires specialized approaches.
The Importance of 3D Cell Models
Traditional two-dimensional (2D) cell cultures, where cells grow as a flat monolayer, have been widely used. However, these simplified models often fail to accurately mimic the complex environment cells experience within a living organism. In a natural setting, cells are surrounded by others and interact with a three-dimensional extracellular matrix (ECM), forming intricate networks that influence their behavior. This lack of realistic cell-cell and cell-environment interactions in 2D cultures can lead to altered cell morphology, growth patterns, and functions, including changes in gene expression and drug sensitivity.
Three-dimensional (3D) cell models, particularly multicellular spheroids, address many of these limitations. They allow cells to aggregate and grow in a more physiologically relevant arrangement. Spheroids enable cells to maintain their native phenotype and engage in complex cell-cell and cell-ECM interactions, mirroring conditions found in living tissues. These 3D structures develop gradients of oxygen, nutrients, and waste products, characteristic of in vivo environments and influence cellular behavior. By providing a more accurate representation of human tissue, 3D spheroids offer improved predictability for research outcomes.
What is a Spheroid Microscope?
A spheroid microscope is a specialized imaging system designed to observe and analyze three-dimensional cellular structures like spheroids and organoids without damaging them. Its primary purpose is to overcome the challenges of viewing thick, opaque 3D samples that scatter light, which traditional microscopes struggle with. These microscopes penetrate deeper into biological samples, allowing visualization of cells and their interactions within the core of a spheroid.
This type of microscope employs advanced optical techniques to achieve “optical sectioning,” effectively taking thin slices of the 3D sample without physically cutting it. This allows for the reconstruction of a complete three-dimensional image. Minimizing photodamage and phototoxicity to living samples during prolonged observation is important for studying dynamic biological processes over time. By reducing light exposure to only the plane being imaged, these systems help maintain cell viability and physiological relevance.
Imaging Technologies for Spheroids
Imaging spheroids requires advanced microscopy techniques that overcome challenges of light scattering and limited penetration depth in thick 3D samples.
Light Sheet Fluorescence Microscopy (LSFM)
LSFM, also known as Selective Plane Illumination Microscopy (SPIM), is a prominent technique. It illuminates a thin plane of the sample with a laser light sheet, then images that plane perpendicularly. This minimizes light exposure to the rest of the spheroid, reducing phototoxicity and photobleaching. This orthogonal illumination and detection setup allows for fast image acquisition and deep penetration, making it suitable for long-term imaging of live, larger spheroids.
Confocal Microscopy
Confocal microscopy, especially spinning disk confocal microscopy, is another widely used technique. Confocal microscopes use a pinhole to block out-of-focus light, achieving optical sectioning and producing sharp images of specific planes within a 3D sample. Spinning disk variants enhance imaging speed by using multiple pinholes on a rotating disk, allowing parallel image acquisition. This makes spinning disk confocal microscopy suitable for observing fast dynamic processes and constructing 3D models of live cells with high sensitivity.
Optical Clearing Techniques
For very large or dense spheroids, optical clearing techniques are often used. These methods chemically treat the sample to make it more transparent by homogenizing its refractive index and removing light-scattering components. Clearing can significantly increase imaging depth, enabling visualization of innermost cells in spheroids that are typically over 100 µm in diameter. Common clearing agents include aqueous-based solutions like Scale and organic solvent-based solutions such as benzyl alcohol and benzyl benzoate (BABB).
Applications in Scientific Research
Spheroid microscopes and the 3D cell models they visualize have diverse applications across scientific fields, providing insights not possible with 2D cultures.
Drug Discovery and Screening
In drug discovery and screening, these microscopes allow researchers to test the efficacy and toxicity of new compounds in a more realistic 3D environment. This improves the prediction of how drugs will behave in vivo, helping reduce high failure rates in preclinical drug development.
Cancer Biology
In cancer biology, spheroid microscopes study tumor growth, metastasis, and drug resistance. Tumor spheroids mimic the structural organization of in vivo tumors, including gradients of oxygen and nutrients, which influence cell behavior and drug penetration. Researchers can observe how cancer cells interact with their microenvironment in a 3D context, leading to a better understanding of disease progression and response to therapies.
Developmental Biology and Regenerative Medicine
Spheroid microscopes contribute to developmental biology by enabling the observation of organ development and cell differentiation in models like embryoid bodies and neurospheres. These 3D models allow the study of biological processes during embryonic development and tissue formation. In regenerative medicine, these microscopes analyze tissue engineering constructs and study the formation of vascular, cartilage, bone, and neural tissues, providing a platform for developing novel therapies for tissue repair and regeneration.